Chimeric immunomodulatory compounds and methods of using the same-IV

ABSTRACT

The invention provides immunomodulatory compounds and methods for immunomodulation of individuals using the immunomodulatory compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/590,150, filed on Oct. 30, 2006, now U.S. Pat. No. 8,114,418, issuedon Feb. 14, 2012, which is a continuation of U.S. application Ser. No.10/623,371, filed Jul. 18, 2003, which is a continuation-in-partapplication of patent application Ser. No. 10/328,578, now U.S. Pat. No.7,785,610, issued on Aug. 31, 2010, which is a continuation-in-part ofpatent application Ser. No. 10/176,883, now U.S. Pat. No. 7,255,868,issued on Aug. 14, 2007, and Ser. No. 10/177,826, both filed on Jun. 21,2002, both of which claim benefit of provisional patent application No.60/299,883, filed Jun. 21, 2001 and provisional patent application No.60/375,253, filed Apr. 23, 2002. All of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to chimeric immunomodulatory compounds(“CICs”) containing nucleic acid and non-nucleic acid moieties, and tothe use of such compounds to modulate an immune response. The inventionfinds use in the fields of biomedicine and immunology.

BACKGROUND

Reference to a publication in this section should not be construed as anindication that the publication is prior art to the present invention.

The type of immune response generated by infection or other antigenicchallenge can generally be distinguished by the subset of T helper (Th)cells involved in the response. The Th1 subset is responsible forclassical cell-mediated functions such as delayed-type hypersensitivityand activation of cytotoxic T lymphocytes (CTLs), whereas the Th2 subsetfunctions more effectively as a helper for B-cell activation. The typeof immune response to an antigen is generally influenced by thecytokines produced by the cells responding to the antigen. Differencesin the cytokines secreted by Th1 and Th2 cells are believed to reflectdifferent biological functions of these two subsets. See, for example,Romagnani (2000) Ann. Allergy Asthma Immunol. 85:9-18.

The Th1 subset may be particularly suited to respond to viralinfections, intracellular pathogens, and tumor cells because it secretesIL-2 and IFN-γ, which activate CTLs. The Th2 subset may be more suitedto respond to free-living bacteria and helminthic parasites and maymediate allergic reactions, since IL-4 and IL-5 are known to induce IgEproduction and eosinophil activation, respectively. In general, Th1 andTh2 cells secrete distinct patterns of cytokines and so one type ofresponse can moderate the activity of the other type of response. Ashift in the Th1/Th2 balance can result in an allergic response, forexample, or, alternatively, in an increased CTL response.

It has been recognized for some time that a Th1-type immune response canbe induced in mammals by administration of certain immunomodulatorypolynucleotides. The immunomodulatory polynucleotides include sequencesreferred to as immunostimulatory sequences (“ISS”), often including aCG. See, e.g., PCT Publications WO 98/55495, WO 97/28259, U.S. Pat. Nos.6,194,388 and 6,207,646; and Krieg et al. (1995) Nature 374:546-49. Formany infectious diseases, such as tuberculosis and malaria, Th2-typeresponses are of little protective value against infection.Protein-based vaccines typically induce Th2-type immune responses,characterized by high titers of neutralizing antibodies but withoutsignificant cell-mediated immunity. Moreover, some types of antibodyresponses are inappropriate in certain indications, most notably inallergy where an IgE antibody response can result in anaphylactic shock.

In view of the need for improved methods of immunotherapy, a need existsfor identification of compounds for modulation of an immune response.

BRIEF SUMMARY OF THE INVENTION

In an aspect, the invention is directed to a chimeric compound havingimmunomodulatory activity. The chimeric immunomodulatory compound(“CIC”) generally comprises one or more nucleic acid moieties and one ormore non-nucleic acid moieties. The nucleic acid moieties in a CIC withmore than one nucleic acid moiety may be the same or different. Thenon-nucleic acid moieties in a CIC with more than one non-nucleic acidmoiety may be the same or different. Thus, in one embodiment the CICcomprises two or more nucleic acid moieties and one or more non-nucleicacid spacer moieties, where at least one non-nucleic acid spacer moietyis covalently joined to two nucleic acid moieties. In an embodiment, atleast one nucleic acid moiety comprises the sequence 5′-CG-3′. In anembodiment, at least one nucleic acid moiety comprises the sequence5′-TCG-3′.

In one aspect, the invention provides a chimeric immunomodulatorycompound that has a core structure with the formula “N₁—S₁—N₂”, where N₁and N₂ are nucleic acid moieties and S₁ is a non-nucleic acid spacermoiety, and where the CIC exhibits immunomodulatory activity. In oneembodiment, the core structure is “N₁—S₁—N₂—S₂—N₃”, where N₃ is anucleic acid moiety and S₂ is a non-nucleic acid spacer moiety. In oneembodiment, the CIC has the core structure“N₁—S₁—N₂—S₂—[N_(v)—S_(v)]_(A)”, where A is an integer between 1 and100, and [N_(v)—S_(v)]_(A) indicates A additional iterations of nucleicacid moieties conjugated to non-nucleic acid spacer moieties, where Sand N are independently selected in each iteration of “[N_(v)—S_(v)]”.In an embodiment, A is at least 2, and at least 4 nucleic acid moietiesin the CIC have different sequences.

In an aspect, the CIC comprises a core structure with the formulaN₁—S₁—N₂ or N₁—S₁—N₂—S₂—N₃ (wherein N₁, N₂, and N₃ are nucleic acidmoieties, S₁ and S₂ are non-nucleic acid spacer moieties, and S₁ and S₂are covalently bound to exactly two nucleic acid moieties). Examples ofsuch CICs are CICs with core structures of the formula (5′—N₁-3′)-S₁—N₂.In one embodiment, N₁ has the sequence 5′-TCGAX-3′, wherein X is 0 to 20nucleotide bases (SEQ ID NO:1). In one embodiment, X is 0 to 3nucleotide bases. In one embodiment, X is CGT. In another embodiment N₁has the sequence 5′-TCGTCGA-3′. In an embodiment, the CIC has thestructure N₁—S₁—N₂—S₂—[N_(v)—S_(v)]_(A) (wherein A is an integer between1 and 100, and [N_(v)—S_(v)]_(A) indicates “A” additional iterations ofnucleic acid moieties conjugated to non-nucleotide spacer moieties,where S and N are independently selected in each iteration of[N_(v)—S_(v)]. In an embodiment, A is 1 to 3.

In another aspect, the invention provides a CIC that has a corestructure with the formula [N_(v)]_(A)---S_(p), where S_(p) is amultivalent spacer covalently bonded to the quantity “A” independentlyselected nucleic acid moieties, N_(v), where A is at least 3, and wherethe CIC exhibits immunomodulatory activity. In one embodiment, the CIChas the core sequence [S_(v)—N_(v)]_(A)---S_(p) where S_(p) is amultivalent spacer covalently bonded to the quantity “A” independentlyselected elements [S_(v)—N_(v)], and independently selected element[S_(v)—N_(v)] includes a spacer moiety covalently bound to a nucleicacid moiety, and wherein A is at least 3. In one embodiment, A is from 3to 50. In a different embodiment, A is greater than 50. In anembodiment, at least 2, at least 3 or at least 4 nucleic acid moietiesin the CIC have different sequences.

In an aspect, the CIC comprises a core structure with the formula[N_(v)]_(A)—S_(p) or [S_(v)—N_(v)]_(A)—S_(p) (where S_(p) is amultivalent spacer covalently bonded to the quantity “A” independentlyselected nucleic acid moieties, N_(v), or independently selectedelements [S_(v)—N_(v)], each independently selected element[S_(v)—N_(v)] comprising a spacer moiety covalently bound to a nucleicacid moiety, wherein A is at least 3. In embodiments, A is from 3 toabout 50 or from about 50 to about 500. In an embodiment, S_(p)comprises a dendrimer. In an embodiment, a nucleic acid moiety of theCIC has a sequence selected from TCGXXXX, TCGXXXX, XTCGXXX, XTCGAXX,TCGTCGA, TCGACGT, TCGAACG, TCGAGAT, TCGACTC, TCGAGCG, TCGATTT, TCGCTTT,TCGGTTT, TCGTTTT, TCGTCGT, ATCGATT, TTCGTTT, TTCGATT, ACGTTCG, AACGTTC,TGACGTT, TGTCGTT, TCGXXX, TCGAXX, TCGTCG, AACGTT, ATCGAT, GTCGTT,GACGTT, TCGXX, TCGAX, TCGAT, TCGTT, TCGTC, TCGA, TCGT, TCGX, or TCG(where “X” is any nucleotide).

In another aspect, the invention provides a CIC that has a corestructure with the formula “N₁—S₁”, where N₁ is a nucleic acid moietyand S₁ is a non-nucleic acid spacer moiety, and the CIC exhibitsimmunomodulatory activity.

The CIC may comprise non-nucleotide spacer moieties comprising, forexample, triethylene glycol, hexaethylene glycol, a polymer comprisingphosphodiester and/or phosphorothioate linked oligoethylene glycolmoieties, C₂-C10 alkyl (e.g., propyl, butyl, hexyl), glycerol or amodified glycerol (e.g., glycerol derivatized at the 1, 2 or 3hydroxy-position; e.g., by addition of an alkylether), pentaerythritolor modified pentaerythritol (pentaerythritol modified at any hydroxyposition(s), e.g., “trebler”), 2-(hydroxymethyl)ethyl,1,3-diamino-2-propanol or modified 1,3-diamino-2-propanol (e.g.,“symmetrical doubler” [Glen Research]), an abasic nucleotide, apolysaccharide (e.g., a cross-linked polysaccharide), a dendrimer,and/or other spacer moiety components disclosed herein, in variouscombinations.

In a related aspect, the invention provides a CIC that is not a branchedCIC and which includes two nucleic acid moieties that are at leastpartially complementary to each other and able to form a duplexstructure. In exemplary embodiments, at least one of the nucleic acidmoieties includes the sequence 5′-TCG-3′,5′-TCGA-3′, 5′-TCGACGT-3′ or5′-TCGTCGA-3, optionally in the 5-prime position.

In a related aspect, the invention provides a branched CIC that has afork structure, an H structure, a comb structure, or a central spacerstructure. In specific embodiments, a non-nucleic acid spacer moiety ofthe CIC includes a glycerol component and/or an oligoethylene glycolcomponent (e.g., HEG). In an embodiment, the non-nucleic acid spacermoiety is a compound spacer. In exemplary embodiments, at least one ofthe nucleic acid moieties includes the sequence 5′-TCG-3′, 5′-TCGA-3′,5′-TCGACGT-3′ or 5′-TCGTCGA-3, optionally in the 5-prime position.

In a related aspect, the invention provides a multimeric CIC including afirst CIC and a second CIC, where the first CIC is not a branched CIC,and the second CIC is or is not a branched CIC, where a nucleic acidmoiety of the first CIC is at least partially complementary to a nucleicacid moiety of the second CIC, and where the two nucleic acid moietiesform a duplex structure. In an embodiment, both the first and secondCICs is a branched CIC. In an embodiment, one or both the first andsecond CIC has a fork structure, an H structure, a comb structure, or acentral spacer structure. In an embodiment, the multimeric CIC has acentral axis structure or a cage structure. In exemplary embodiments, atleast one nucleic acid moiety in one or more of the CICs of themultimeric CIC includes the sequence 5′-TCG-3′, 5′-TCGA-3′,5′-TCGACGT-3′ or 5′-TCGTCGA-3, optionally in the 5-prime position. In anembodiment, all of the nucleic acid moieties, or all of 5-primemoieties, in one, two or more of the CICs of the multimeric CIC have thesame sequence.

In various embodiments, a CIC described above has one or more of thefollowing characteristics: (i) the CIC includes at least one nucleicacid moiety less than 8 nucleotides (or base pairs) in length, or,alternatively, less than 7 nucleotides in length (ii) all of the nucleicacid moieties of the CIC are less than 8 nucleotides in length, or,alternatively, less than 7 nucleotides in length, (iii) the CIC includesat least one nucleic acid moiety that includes the sequence 5′-CG-3′(e.g., 5′-TCG-3′), (iv) the CIC includes at least two nucleic acidmoieties having different sequences, (v) all of the nucleic acidmoieties of the CIC have the same sequence, (vi) the CIC includes atleast one non-nucleic acid spacer moiety that is or comprisestriethylene glycol, hexaethylene glycol, propyl, butyl, hexyl, glycerolor a modified glycerol (e.g., glycerol derivatized at the 1, 2 or 3hydroxy-position; e.g., by addition of an alkylether), pentaerythritolor modified pentaerythritol (pentaerythritol modified at any hydroxyposition(s), e.g., “trebler”), 2-(hydroxymethyl)ethyl,1,3-diamino-2-propanol or modified 1,3-diamino-2-propanol (e.g.,“symmetrical doubler” [Glen Research]), an abasic nucleotide, apolysaccharide (e.g., a cross-linked polysaccharide), or a dendrimer.

In various embodiments, a CIC described herein has one or more of thefollowing characteristics: (vii) the CIC includes at least one nucleicacid moiety of the CIC that does not have “isolated immunomodulatoryactivity,” (viii) the CIC does not include any nucleic acid moiety with“isolated immunomodulatory activity,” (ix) the CIC includes at least onenucleic acid moiety of the CIC that has “inferior isolated immunologicalactivity.” “Isolated immunomodulatory activity” and “inferior isolatedimmunological activity” are described herein. In various embodiments aCIC described herein includes at least one nucleic acid moiety that isdouble-stranded or partially double-stranded. CICs can be designed withself-complementary nucleic acid moieties such that duplexes can beformed. See, e.g., C-84, C-85, and C-87.

Thus, in various aspects, the invention provides a CIC comprising two ormore nucleic acid moieties and one or more non-nucleic acid spacermoieties, wherein at least one spacer moiety is covalently joined to twonucleic acid moieties and at least one nucleic acid moiety comprises thesequence 5′-CG-3′, and wherein said CIC has immunomodulatory activity.The CIC may comprise at least three nucleic acid moieties, wherein eachnucleic acid moiety is covalently joined to at least one non-nucleicacid spacer moiety. The CIC may have at least one immunomodulatoryactivity such as (a) the ability to stimulate IFN-γ production by humanperipheral blood mononuclear cells; (b) the ability to stimulate IFN-αproduction by human peripheral blood mononuclear cells; and/or (c) theability to stimulate proliferation of human B cells.

One or more nucleic acid moieties of the CIC can comprise a sequencesuch as 5′-TCGA-3′, 5′-TCGACGT-3′, 5′-TCGTCGA-3′ and 5′-ACGTTCG-3′. Inan embodiment, one or more nucleic acid moieties of the CIC can have thesequence 5′-X₁X₂CGX₃X₄-3′ (where X₁ is zero to ten nucleotides; X₂ isabsent or is A, T, or U; X₃ is absent or is A; and X₄ is zero to tennucleotides, and wherein the nucleic acid moiety is conjugated to aspacer moiety, for example at the 3′ end). In an embodiment, the sum ofnucleotides in X₁, X₂, X₃, and X₄ can be less than 8, less than 7, lessthan 6, less than 5 or less than 4. In some embodiments, one or morenucleic acid moieties of the CIC can have a nucleic acid sequence suchas TCGXXXX, TCGAXXX, XTCGXXX, XTCGAXX, TCGTCGA, TCGACGT, TCGAACG,TCGAGAT, TCGACTC, TCGAGCG, TCGATTT, TCGCTTT, TCGGTTT, TCGTTTT, TCGTCGT,ATCGATT, TTCGTTT, TTCGATT, ACGTTCG, AACGTTC, TGACGTT, TGTCGTT, TCGXXX,TCGAXX, TCGTCG, AACGTT, ATCGAT, GTCGTT, GACGTT, TCGXX, TCGAX, TCGAT,TCGTT, TCGTC, TCGA, TCGT, TCGX, or TCG (where “X” is any nucleotide).

In one embodiment, one or more nucleic acid moieties comprises 3 to 7bases. In one embodiment, the nucleic acid moiety comprises 3 to 7 basesand has the sequence 5′-[(X)₀₋₂]TCG[(X)₂₋₄]-3′, or 5′-TCG[(X)₂₋₄]-3′, or5′-TCG(A/T)[(X)₁₋₃]-3′, or 5′-TCG(A/T)CG(A/T)-3′, or 5′-TCGACGT-3′ or5′-TCGTCGA-3′, wherein each X is an independently selected nucleotide.In some embodiments, the CIC contains at least 3, at least 10, at least30 or at least 100 nucleic acid moieties having a sequence describedabove.

In one aspect, the invention provides a chimeric immunomodulatorycompound (CIC) that stimulates production of IFN-α from human peripheralblood mononuclear cells and has at least three nucleic acid moieties andat least one nonnucleic acid spacer moiety, where at least one nucleicacid moiety comprises a motif 5′-TCGXCGX 5′-TCGXTCG5′-TCGXXCG, or5′-TCGCGXX, where X is any nucleotide (for example, 5′^(F)-TCGXCGX5′^(F)-TCGXTCG, 5′^(F)-TCGXXCG, or 5′^(F)-TCGCGXX). The CIC may have anyof the structures described herein for CICs and, for example, maycomprise at least one multivalent nonnucleic acid spacer moiety and/ormay comprise at least one nonnucleic acid spacer moiety comprising HEG,TEG, propyl, butyl, hexyl, pentaerythritol, 2-(hydroxymethyl)ethyl,glycerol, a polysaccharide, 1,3-diamino-2-propanol, or a dendrimer.

The CIC can include at least one nucleic acid moiety that is less than 8nucleotides in length. Optionally all the nucleic acid moieties in theCIC are less than 8 nucleotides in length. In some embodiments, all thenucleic acid moieties in the CIC that comprise the sequence 5′-CG-3′ areless than 8 nucleotides in length. The CIC can include at least 2nucleic acid moieties having different sequences. The CIC can contain atleast one nucleic acid moiety does not comprise the sequence 5′-CG-3′.The CIC may include at least one nucleic acid moiety that does not haveisolated immunological activity or has inferior isolated immunologicalactivity. Optionally no nucleic acid moiety of the CIC has isolatedimmunomodulatory activity. The linkages between the nucleotides of thenucleic acid moieties may include phosphodiester, phosphorothioateester, phosphorodithioate ester, other covalent linkages, and mixturesthereof. Similarly, the linkages between nucleic acid moieties andspacer moieties or between components of spacer moieties may includephosphodiester, phosphorothioate ester, phosphorodithioate ester, otherlinkages, and mixtures thereof.

In an embodiment, the CIC includes a reactive linking group (e.g., areactive thio group). The CIC may be linked or noncovalently associatedwith a polypeptide, e.g., a polypeptide antigen.

The invention also provides compositions comprising a CIC along with apharmaceutically acceptable excipient and/or an antigen and/or acationic microcarrier (such as a polymer of lactic acid and glycolicacid). The composition can be essentially endotoxin-free.

In an aspect, the invention provides a composition containing a CICdescribed herein and a pharmaceutically acceptable excipient, an antigen(e.g., an antigen to which an immune response is desired), or both. Inan embodiment, the composition is formulated under GMP standards. In anembodiment, the composition is prepared by a process that includesassaying the composition for the presence of endotoxin. In anembodiment, the composition is essentially endotoxin-free. In anembodiment, the composition does not contain liposomes.

In an aspect, the invention provides the use of a CIC as describedherein for the manufacture of a medicament.

In an aspect, the invention provides a method of modulating an immuneresponse of a cell by contacting the cell with a CIC-containingcomposition. In an embodiment, the CIC-containing composition comprisesa multimeric CIC.

In an aspect, the invention provides a method of modulating an immuneresponse in an individual by administering a chimeric immunomodulatorycompound or CIC-containing composition as described herein, in an amountsufficient to modulate an immune response in the individual. In oneembodiment, the individual suffers from a disorder associated with aTh2-type immune response, for example, an allergy or allergy-inducedasthma. In one embodiment, the individual has an infectious disease.

In an aspect, the invention provides a method of increasinginterferon-gamma (IFN-γ) in an individual by administering a CIC orcomposition as described herein, in an amount sufficient to increaseIFN-γ in the individual. In an embodiment, the individual has aninflammatory disorder. In an embodiment, the individual has idiopathicpulmonary fibrosis.

In an aspect, the invention provides a method of increasinginterferon-alpha (IFN-α) in an individual, by administering a CIC orcomposition as described herein, in an amount sufficient to increaseIFN-α in the individual. In an embodiment, the individual has a viralinfection.

In one aspect, the invention provides a CIC that stimulates productionof IFN-α from human peripheral blood mononuclear cells but does notstimulate human B cell proliferation, or stimulates little B cellproliferation. For example, but not limitation, this CIC may comprise anucleic acid moiety comprising the sequence 5′-TCGAX_(N), (for example,5′^(F)-TCGAX_(N)) where X is any nucleotide and n is 1, 2, or 3; anucleic acid moiety comprising the sequence 5′-TCGAX_(N), where X is anyamino acid and n is an integer from 4 to 9; or a nucleic acid moietycomprising the sequence 5′-TCGACGX_(N), (for example,5′^(F)-TCGACGX_(N)) where X is any nucleotide and n is 1 or,alternatively, n is 2, 3, or an integer from 4 to 7. In an embodiment, Xis T (5′-TCGACGT). For example, but not limitation, the CIC may have thestructure (5′-TCGACGT-HEG)₂-glycerol-HEG-5′-TCGACGT or(5′-TCGACGT-HEG)₃-trebler-HEG-5′-TCGACGT.

In a related aspect, the invention provides a composition comprising aCIC that stimulates production of IFN-α from human peripheral bloodmononuclear cells but does not stimulate human B cell proliferation, orstimulates little human B cell proliferation, and a pharmaceuticallyacceptable excipient. In related embodiments the composition alsoincludes an antigen and/or a cationic microsphere (e.g. as describedherein).

In an aspect, the invention provides a method of ameliorating a symptomof an infectious disease in an individual, by administering an effectiveamount of a CIC or composition, as described herein, to the individual,where the effective amount is an amount sufficient to ameliorate asymptom of the infectious disease.

In an aspect, the invention provides a method of ameliorating anIgE-related disorder in an individual, by administering an effectiveamount of a CIC or composition described herein to an individual havingan IgE-related disorder, where an effective amount is an amountsufficient to ameliorate a symptom of the IgE-related disorder. In anembodiment, the IgE-related disorder is allergy or an allergy-relateddisorder.

The invention further provides a method of modulating an immune responsein an individual by administering to an individual a CIC in an amountsufficient to modulate an immune response in said individual. Inembodiments, the individual has cancer and/or suffers from a disorderassociated with a Th2-type immune response (e.g., an allergy orallergy-induced asthma) and/or has an infectious disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure of certain reagents useful for synthesis ofnon-nucleic acid spacer moieties of CICs. Shown aredimethoxytrityl-protected phosphoramidite spacer moiety precursors forHEG, propyl, TEG, HME, butyl, and abasic spacer moieties.

FIG. 2 shows the structure of certain reagents useful for synthesis ofsymmetric or asymmetric non-nucleic acid spacer moieties of CICs. Shownare dimethoxytrityl-protected phosphoramidite spacer moiety precursorsfor glycerol [2] “symmetrical branched”), levulinyl-glycerol [3](“asymmetrical branched”), “trebler” [9] and “symmetrical doubler” [10]spacer moieties.

FIGS. 3A and 3B diagram the synthesis of a branched CIC.

FIG. 4 shows the synthetic scheme for C-105.

FIG. 5 shows induction of immune-associated genes in the mouse lungafter intranasal treatment with CICs.

FIGS. 6A-C show the effect of CICs on levels of IL-12 p40 (FIG. 6A),IL-6 (FIG. 6B), and TNF-alpha (FIG. 6C).

FIGS. 7A-B show the structures of C-8 (FIG. 7A) and C-101 (FIG. 7B).

FIGS. 8A-8H show examples of CICs having defined secondary or tertiarystructure. FIG. 8A shows a linear CIC having the structure of a hairpinduplex; FIG. 8B shows a branched CIC having a “fork” structure; FIG. 8Cshows a branched CIC with an “H” structure; FIG. 8D shows a branched CICwith a “comb” structure; FIG. 8E shows a branched CIC with a“central-spacer” structure; FIG. 8F shows a branched CIC with a“central-spacer” structure; FIG. 8G illustrates synthesis of a branchedCIC with a “central-spacer” structure by a conjugation strategy; FIG. 8Hshows a CIC dendrimer. (H=HEG; N=1-5; A=5′ adenosine; G=5′ guanosine;“|” indicates base-pairing). The sequence identifier for the sequenceshown in FIG. 8A is ATCGATCGTTCGAGCGAC (SEQUENCE ID NO:140).

FIGS. 9A-9G show examples of CIC multimers. FIG. 9A shows a CIC multimerhaving the structure of a linear CIC duplex and comprising two identicalCICs; FIG. 9B shows a CIC multimer having the structure of a linear CICduplex and comprising two different CICs; FIG. 9C shows a linear dimerhaving 5′ ends that are not base-paired; FIG. 9D shows a CIC multimerhaving the structure of a concatamer of five linear CICs; FIG. 9E showsa CIC multimer with a “central axis” structure; FIG. 9F shows a CICmultimer with a “cage” structure; FIG. 9G shows a CIC multimer with a“starfish” structure. (H=HEG; A=5′ adenosine; T=5′ thymidine; G=5′guanosine). The sequence identifiers for sequences shown in FIGS. 9A-9Gare: ATCGATCGTTCGAGCGAC (SEQ ID NO:140); GTCGCTCGAACGATCGAT (SEQ IDNO:141); AGGGTTTTTTTTTITITT(SEQ ID NO:142);TCGATCGATCGATCGTTCGAGCGAC(SEQ ID NO:143);GTCGCTCGAACGATCGATTTAACAAAC(SEQ ID NO:144);GTCGCTCGAACGATCGATAATAAAT(SEQ ID NO:145); TCGATCGTTATCGATCGTTCGAGCGAC(SEQ ID NO:146); TCGATTCGAGCG (SEQ ID NO:147);TCGTTCGAGCGAATTCGCTCGAACGATCTT (SEQ ID NO:148); TCGTTTTTTTTCGC (SEQ IDNO:149); AAAAAAAACGCCG (SEQ ID NO:150); TCGCGAAAAAAAACGA (SEQ IDNO:151); ATCATCCGAACGTTGA(SEQ ID NO:152).

FIG. 10 shows effects of CIC structure, spacer composition, and NAMsequence on IFN-α and IFN-γ production. PBMCs were isolated from 8donors and stimulated with 20 μg/ml P-6 or CIC for 24 h. Cell-freesupernatants were assayed for IFN-γ, and IFN-α content by ELISA. Dataare shown as means±SEM. Statistical relevance: **, p<0.01, *, p<0.05,where P-6 and the linear CICs (C-74, C-75, C-76, C-77, C-73, C-41, C-21and C-51) were compared to the linear chimeric control ODN, M-2 andbranched CICs (C-143, C-94, C-101, C-142, C-103, C-104, C-28, C-145,C-156, C-157, C-158, and C-125) were compared to the branched chimericcontrol ODN, M-3.

FIG. 11 shows the effect of NAM sequence (motifs) on the level of humanB cell activity. Purified human B cells from 2 donors were stimulatedwith 5 μg/ml P-6 or CIC for 96 h. Proliferation was assessed by³H-thymidine incorporation. This assay is representative of two separateassays with two donors each.

DETAILED DESCRIPTION OF THE INVENTION

I. General Methods

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry,nucleic acid chemistry, and immunology, which are within the skill ofthe art. Such techniques are explained fully in the literature, such as,Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al.,1989) and Molecular Cloning: A Laboratory Manual, third edition(Sambrook and Russel, 2001), (jointly and individually referred toherein as “Sambrook”). Oligonucleotide Synthesis (M. J. Gait, ed.,1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Handbook ofExperimental Immunology (D. M. Weir & C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds.,1987); Current Protocols in Molecular Biology (F. M. Ausubel et al.,eds., 1987, including supplements through 2001); PCR: The PolymeraseChain Reaction, (Mullis et al., eds., 1994); Current Protocols inImmunology (J. E. Coligan et al., eds., 1991); The Immunoassay Handbook(D. Wild, ed., Stockton Press NY, 1994); Bioconjugate Techniques (GregT. Hermanson, ed., Academic Press, 1996); Methods of ImmunologicalAnalysis (R. Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim:VCH Verlags gesellschaft mbH, 1993), Harlow and Lane (1988) Antibodies,A Laboratory Manual, Cold Spring Harbor Publications, New York, andHarlow and Lane (1999) Using Antibodies: A Laboratory Manual Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (jointly andindividually referred to herein as “Harlow and Lane”), Beaucage et al.eds., Current Protocols in Nucleic Acid Chemistry John Wiley & Sons,Inc., New York, 2000); and Agrawal, ed., Protocols for Oligonucleotidesand Analogs, Synthesis and Properties Humana Press Inc., New Jersey,1993).

II. Definitions

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless otherwise indicated or clear from context. Forexample, as will be apparent from context, “a” chimericimmunomodulatory/immunostimulatory compound (“CIC”) can include one ormore CICs. Similarly, reference in the singular form of a componentelement of a CIC (i.e., nucleic acid moiety or non-nucleic acid spacermoiety) can include multiple elements. For example, a description of “anucleic acid moiety” in a CIC can also describe two or more “nucleicacid moieties” in the CIC.

As used interchangeably herein, the terms “polynucleotide,”“oligonucleotide” and “nucleic acid” include single-stranded DNA(ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) anddouble-stranded RNA (dsRNA), modified oligonucleotides andoligonucleosides, or combinations thereof. The nucleic acid can belinearly or circularly configured, or the oligonucleotide can containboth linear and circular segments. Nucleic acids are polymers ofnucleosides joined, e.g., through phosphodiester linkages or alternatelinkages, such as phosphorothioate esters. A nucleoside consists of apurine (adenine (A) or guanine (G) or derivative thereof) or pyrimidine(thymine (T), cytosine (C) or uracil (U), or derivative thereof) basebonded to a sugar. The four nucleoside units (or bases) in DNA arecalled deoxyadenosine, deoxyguanosine, deoxythymidine, anddeoxycytidine. A nucleotide is a phosphate ester of a nucleoside.

The term “3′” generally refers to a region or position in apolynucleotide or oligonucleotide 3′ (downstream) from another region orposition in the same polynucleotide or oligonucleotide.

The term “5′” generally refers to a region or position in apolynucleotide or oligonucleotide 5′ (upstream) from another region orposition in the same polynucleotide or oligonucleotide.

An element, e.g., region, portion, non-nucleic acid spacer moiety,nucleic acid moiety, or sequence is “adjacent” to another element, e.g.,region, portion, non-nucleic acid spacer moiety, nucleic acid moiety, orsequence, when it directly abuts that region, portion, spacer orsequence.

The term “CIC-antigen conjugate” refers to a complex in which a CIC andan antigen are linked. Such conjugate linkages include covalent and/ornon-covalent linkages.

The term “antigen” means a substance that is recognized and boundspecifically by an antibody or by a T cell antigen receptor. Antigenscan include peptides, proteins, glycoproteins, polysaccharides, complexcarbohydrates, sugars, gangliosides, lipids and phospholipids; portionsthereof and combinations thereof. The antigens can be those found innature or can be synthetic. Antigens suitable for administration with aCIC includes any molecule capable of eliciting a B cell or T cellantigen-specific response. Preferably, antigens elicit an antibodyresponse specific for the antigen. Haptens are included within the scopeof “antigen.” A hapten is a low molecular weight compound that is notimmunogenic by itself but is rendered immunogenic when conjugated withan immunogenic molecule containing antigenic determinants. Smallmolecules may need to be haptenized in order to be rendered antigenic.Preferably, antigens of the present invention include peptides, lipids(e.g. sterols, fatty acids, and phospholipids), polysaccharides such asthose used in Hemophilus influenza vaccines, gangliosides andglycoproteins.

“Adjuvant” refers to a substance which, when added to an immunogenicagent such as antigen, nonspecifically enhances or potentiates an immuneresponse to the agent in the recipient host upon exposure to themixture.

The term “peptide” are polypeptides that are of sufficient length andcomposition to effect a biological response, e.g., antibody productionor cytokine activity whether or not the peptide is a hapten. Typically,the peptides are at least six amino acid residues in length. The term“peptide” further includes modified amino acids (whether or notnaturally or non-naturally occurring), such modifications including, butnot limited to, phosphorylation, glycosylation, pegylation, lipidizationand methylation.

“Antigenic peptides” can include purified native peptides, syntheticpeptides, recombinant peptides, crude peptide extracts, or peptides in apartially purified or unpurified active state (such as peptides that arepart of attenuated or inactivated viruses, cells, micro-organisms), orfragments of such peptides. An “antigenic peptide” or “antigenpolypeptide” accordingly means all or a portion of a polypeptide whichexhibits one or more antigenic properties. Thus, for example, an “Amb a1 antigenic polypeptide” or “Amb a 1 polypeptide antigen” is an aminoacid sequence from Amb a 1, whether the entire sequence, a portion ofthe sequence, and/or a modification of the sequence, which exhibits anantigenic property (i.e., binds specifically to an antibody or a T cellreceptor).

A “delivery molecule” or “delivery vehicle” is a chemical moiety whichfacilitates, permits, and/or enhances delivery of a CIC, CIC-antigenmixture, or CIC-antigen conjugate to a particular site and/or withrespect to particular timing. A delivery vehicle may or may notadditionally stimulate an immune response.

An “allergic response to antigen” means an immune response generallycharacterized by the generation of eosinophils (usually in the lung)and/or antigen-specific IgE and their resultant effects. As iswell-known in the art, IgE binds to IgE receptors on mast cells andbasophils. Upon later exposure to the antigen recognized by the IgE, theantigen cross-links the IgE on the mast cells and basophils causingdegranulation of these cells, including, but not limited, to histaminerelease. It is understood and intended that the terms “allergic responseto antigen”, “allergy”, and “allergic condition” are equally appropriatefor application of some of the methods of the invention. Further, it isunderstood and intended that the methods of the invention include thosethat are equally appropriate for prevention of an allergic response aswell as treating a pre-existing allergic condition.

As used herein, the term “allergen” means an antigen or antigenicportion of a molecule, usually a protein, which elicits an allergicresponse upon exposure to a subject. Typically the subject is allergicto the allergen as indicated, for instance, by the wheal and flare testor any method known in the art. A molecule is said to be an allergeneven if only a small subset of subjects exhibit an allergic (e.g., IgE)immune response upon exposure to the molecule. A number of isolatedallergens are known in the art. These include, but are not limited to,those provided in Table 1 herein.

The term “desensitization” refers to the process of the administrationof increasing doses of an allergen to which the subject has demonstratedsensitivity. Examples of allergen doses used for desensitization areknown in the art, see, for example, Fornadley (1998) Otolaryngol. Clin.North Am. 31:111-127.

“Antigen-specific immunotherapy” refers to any form of immunotherapywhich involves antigen and generates an antigen-specific modulation ofthe immune response. In the allergy context, antigen-specificimmunotherapy includes, but is not limited to, desensitization therapy.

The term “microcarrier” refers to a particulate composition which isinsoluble in water and which has a size of less than about 150, 120 or100 μm, more commonly less than about 50-60 μm, and may be less thanabout 10 μm or even less than about 5 μm. Microcarriers include“nanocarriers”, which are microcarriers have a size of less than about 1μm, preferably less than about 500 nm. Microcarriers include solid phaseparticles such a particles formed from biocompatible naturally occurringpolymers, synthetic polymers or synthetic copolymers, althoughmicrocarriers formed from agarose or cross-linked agarose may beincluded or excluded from the definition of microcarriers herein as wellas other biodegradable materials known in the art. Solid phasemicrocarriers are formed from polymers or other materials which arenon-erodible and/or non-degradable under mammalian physiologicalconditions, such as polystyrene, polypropylene, silica, ceramic,polyacrylamide, gold, latex, hydroxyapatite, and ferromagnetic andparamagnetic materials. Biodegradable solid phase microcarriers may beformed from polymers which are degradable (e.g., poly(lactic acid),poly(glycolic acid) and copolymers thereof, such as poly(D,L-lactide-co-glycolide) or erodible (e.g., poly(ortho esters such as3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5] undecane (DETOSU) orpoly(anhydrides), such as poly(anhydrides) of sebacic acid) undermammalian physiological conditions. Microcarriers are typicallyspherical in shape, but microcarriers which deviate from spherical shapeare also acceptable (e.g., ellipsoidal, rod-shaped, etc.). Due to theirinsoluble nature, solid phase microcarriers are filterable from waterand water-based (aqueous) solutions (e.g., using a 0.2 micron filter).Microcarriers may also be liquid phase (e.g., oil or lipid based), suchas liposomes, iscoms (immune-stimulating complexes, which are stablecomplexes of cholesterol, phospholipid and adjuvant-active saponin)without antigen, or droplets or micelles found in oil-in-water orwater-in-oil emulsions. Biodegradable liquid phase microcarrierstypically incorporate a biodegradable oil, a number of which are knownin the art, including squalene and vegetable oils. The term“nonbiodegradable”, as used herein, refers to a microcarrier which isnot degraded or eroded under normal mammalian physiological conditions.Generally, a microcarrier is considered nonbiodegradable if it notdegraded (i.e., loses less than 5% of its mass or average polymerlength) after a 72 hour incubation at 37° C. in normal human serum.

A microcarrier is considered “biodegradable” if it is degradable orerodable under normal mammalian physiological conditions. Generally, amicrocarrier is considered biodegradable if it is degraded (i.e., losesat least 5% of its mass or average polymer length) after a 72 hourincubation at 37° C. in normal human serum.

The term “CIC/microcarrier complex” or “CIC/MC complex” refers to acomplex of a CIC and a microcarrier. The components of the complex maybe covalently or non-covalently linked. Non-covalent linkages may bemediated by any non-covalent bonding force, including by hydrophobicinteraction, ionic (electrostatic) bonding, hydrogen bonds and/or vander Waals attractions. In the case of hydrophobic linkages, the linkageis generally via a hydrophobic moiety (e.g., cholesterol) covalentlylinked to the CIC.

An “individual” or “subject” is a vertebrate, such as avian, preferablya mammal, such as a human. Mammals include, but are not limited to,humans, non-human primates, farm animals, sport animals, experimentalanimals, rodents (e.g., mice and rats) and pets.

An “effective amount” or a “sufficient amount” of a substance is thatamount sufficient to effect a desired biological effect, such asbeneficial results, including clinical results, and, as such, an“effective amount” depends upon the context in which it is beingapplied. In the context of administering a composition that modulates animmune response to a co-administered antigen, an effective amount of aCIC and antigen is an amount sufficient to achieve such a modulation ascompared to the immune response obtained when the antigen isadministered alone. An effective amount can be administered in one ormore administrations.

The term “co-administration” as used herein refers to the administrationof at least two different substances sufficiently close in time tomodulate an immune response. Preferably, co-administration refers tosimultaneous administration of at least two different substances.

“Stimulation” of an immune response, such as Th1 response, means anincrease in the response, which can arise from eliciting and/orenhancement of a response. Similarly, “stimulation” of a cytokine orcell type (such as CTLs) means an increase in the amount or level ofcytokine or cell type.

An “IgE associated disorder” is a physiological condition which ischaracterized, in part, by elevated IgE levels, which may or may not bepersistent. IgE associated disorders include, but are not limited to,allergy and allergic reactions, allergy-related disorders (describedbelow), asthma, rhinitis, atopic dermatitis, conjunctivitis, urticaria,shock, Hymenoptera sting allergies, food allergies, and drug allergies,and parasite infections. The term also includes related manifestationsof these disorders. Generally, IgE in such disorders isantigen-specific.

An “allergy-related disorder” means a disorder resulting from theeffects of an antigen-specific IgE immune response. Such effects caninclude, but are not limited to, hypotension and shock. Anaphylaxis isan example of an allergy-related disorder during which histaminereleased into the circulation causes vasodilation as well as increasedpermeability of the capillaries with resultant marked loss of plasmafrom the circulation. Anaphylaxis can occur systemically, with theassociated effects experienced over the entire body, and it can occurlocally, with the reaction limited to a specific target tissue or organ.

The term “viral disease”, as used herein, refers to a disease which hasa virus as its etiologic agent. Examples of viral diseases includehepatitis B, hepatitis C, influenza, acquired immunodeficiency syndrome(AIDS), and herpes zoster.

As used herein, and as well-understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation or amelioration ofone or more symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, preventing spread of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.

“Palliating” a disease or disorder means that the extent and/orundesirable clinical manifestations of a disorder or a disease state arelessened and/or time course of the progression is slowed or lengthened,as compared to not treating the disorder. Especially in the allergycontext, as is well understood by those skilled in the art, palliationmay occur upon modulation of the immune response against an allergen(s).Further, palliation does not necessarily occur by administration of onedose, but often occurs upon administration of a series of doses. Thus,an amount sufficient to palliate a response or disorder may beadministered in one or more administrations.

An “antibody titer”, or “amount of antibody”, which is “elicited” by aCIC and antigen refers to the amount of a given antibody measured at atime point after administration of the CIC and antigen.

A “Th1-associated antibody” is an antibody whose production and/orincrease is associated with a Th1 immune response. For example, IgG2a isa Th1-associated antibody in mouse. For purposes of this invention,measurement of a Th1-associated antibody can be measurement of one ormore such antibodies. For example, in humans, measurement of aTh1-associated antibody could entail measurement of IgG1 and/or IgG3.

A “Th2-associated antibody” is an antibody whose production and/orincrease is associated with a Th2 immune response. For example, IgG1 isa Th2-associated antibody in mouse. For purposes of this invention,measurement of a Th2-associated antibody can be measurement of one ormore such antibodies. For example, in human, measurement of aTh2-associated antibody could entail measurement of IgG2 and/or IgG4.

To “suppress” or “inhibit” a function or activity, such as cytokineproduction, antibody production, or histamine release, is to reduce thefunction or activity when compared to otherwise same conditions exceptfor a condition or parameter of interest, or alternatively, as comparedto another condition. For example, a composition comprising a CIC andantigen which suppresses histamine release reduces histamine release ascompared to, for example, histamine release induced by antigen alone. Asanother example, a composition comprising a CIC and antigen whichsuppresses antibody production reduces extent and/or levels of antibodyas compared to, for example, extent and/or levels of antibody producedby antigen alone.

As used herein manufactured or formulated “under GMP standards,” whenreferring to a pharmaceutical composition means the composition isformulated as sterile, substantially isotonic and in full compliancewith all Good Manufacturing Practice (GMP) regulations of the U.S. Foodand Drug Administration.

As used herein, the term “immunogenic” has the normal meaning in the artand refers to an agent (e.g., polypeptide) that elicits an adaptiveimmune response upon injection into a person or animal. The immuneresponse may be B cell (humoral) and/or T cell (cellular).

All ranges are intended to be inclusive of the terminal values. Thus, apolymer of “from 2 to 7 nucleotides” or “between 2 and 7 nucleotides”includes polymers of 2 nucleotides and polymers of 7 nucleotides. Wherea lower limit and an independently selected upper limit are described,it is understood that the upper limit is higher than the lower limit.

III. Chimeric Immunomodulatory Compounds

The invention provides chimeric immunomodulatory compounds (“CICs”)useful, inter alfa, for modulating an immune response in individualssuch as mammals, including humans. The present invention is based, inpart, on the discovery that some chimeric molecules containing nucleicacid moieties covalently bound to non-nucleic acid spacer moieties haveimmunomodulatory activity, particularly in human cells. Surprisingly,this activity is manifest even in cases in which the nucleic acidmoieties have a sequence that, if presented as an isolatedpolynucleotide, do not exhibit significant immunomodulatory activity.

Thus, the invention provides new reagents and methods for modulating animmune response, including treatment and prophylaxis of disease inhumans and other animals.

The following sections describe the structure and properties of the CICsof the invention, as well as the structure and properties of thecomponent nucleic acid moieties and non-nucleic acid spacer moieties.

1. Core Structure of CIC

CICs of the present invention contain one or more nucleic acid moietiesand one or more non-nucleic acid spacer moieties. CICs having a varietyof structures are contemplated. For illustration, exemplary CICs havecore structures described in formulas I-VIII, below. Formulas I-III showcore sequences for “linear CICs.” Formulas IV-VI show core sequences for“branched CICs.” Formula VIII shows a core structure for “single-spacerCICs.”

In each formula provided below, “N” designates a nucleic acid moiety(oriented in either a 5→3′ or 3→5′ orientation) and “S” designates anon-nucleic acid spacer moiety. A dash (“-”) designates a covalent bondbetween a nucleic acid moiety and a non-nucleic acid spacer moiety. Adouble dash (“--”) designates covalent bonds between a non-nucleic acidspacer moiety and at least 2 nucleic acid moieties. A triple dash(“---”) designates covalent bonds between a non-nucleic acid spacermoiety and multiple (i.e., at least 3) nucleic acid moieties. Subscriptsare used to designate differently positioned nucleic acid or non-nucleicacid spacer moieties. However, the use of subscripts to distinguishdifferent nucleic acid moieties is not intended to indicate that themoieties necessarily have a different structure or sequence. Similarly,the use of subscripts to distinguish different spacer moieties is notintended to indicate that the moieties necessarily have differentstructures. For example, in formula II, infra, the nucleic acid moietiesdesignated N₁ and N₂ can have the same or different sequences, and thespacer moieties designated S₁ and S₂ can have the same or differentstructures.

A. Linear CICs

In one embodiment, the CIC comprises the core structureN₁—S₁—N₂  (I).

In one embodiment, the CIC comprises the core structureN₁—S₁—N₂—S₂—N₃  (II).

In one embodiment, the CIC comprises the core structureN₁—S₁—N₂—S₂—[N_(v)—S_(v)]_(A)  (III)where A is an integer between 1 and about 100 and [N_(v)—S_(v)]indicates A additional iterations of nucleic acid moieties conjugated tonon-nucleic acid spacer moieties. The subscript “v” indicates that N andS are independently selected in each iteration of “[N_(v)—S_(v)].” “A”is sometimes between 1 and about 10, sometimes between 1 and 3,sometimes exactly 1, 2, 3, 4 or 5. In some embodiments, A is an integerin a range defined by a lower limit of 1, 2, 3, 4, or 5, and anindependently selected upper limit of 10, 20, 50 or 100 (e.g., between 3and 10). A non-nucleic acid spacer moiety that is covalently linked toexactly two nucleic acid moieties (e.g., S₁ in structures I-III, supra)can be referred to as a “linear spacer.”

In some embodiments of the invention, the CIC has the structure offormula I, II or III. However, according to the invention, in someembodiments, linear CICs comprise, but are not necessarily limited to,the structures provided in formulas I-III. That is, formulas I, II, andIII define core structures in which the non-nucleic acid spacer moietiesin the core structure are covalently bound to no more than two nucleicacid moieties. However, it is contemplated that, in many embodiments,additional chemical moieties (e.g., phosphate, mononucleotide,additional nucleic acid moieties, alkyl, amino, thio or disulfide groupsor linking groups, and/or spacer moieties) are covalently bound at thetermini of the core structures. For example, if all nucleic acidmoieties in a CIC are 5′-TCGTCGA-3′, and spacer moieties are selectedfrom hexaethylene glycol (“HEG”), a phosphorothioate-linked multimer ofHEG, and glycerol, CICs having a core structure of formula I includeeach of the following formulas:

TCGTCGA-HEG-TCGTCGA-OH (Ia) TCGTCGA-HEG-TCGTCGA-PO₄ (Ib)TCGTCGA-HEG-TCGTCGA-HEG (Ic) HEG-TCGTCGA-HEG-TCGTCGA-HEG (Id)TCGTCGA-HEG-TCGTCGA-HEG-TCGTCGA (Ie) TCGTCGA-HEG-TCGTCGA-(HEG)₄-TCGTCGA(If) (TCGTCGA)₂-glycerol-TCGTCGA-HEG-TCGTCGA (Ig)PO₄-TCGTCGA-HEG-TCGTCGA (Ih) TCGTCGA-(HEG)₁₅-T (Ii)(TCGTCGA-HEG)₂-glycerol-HEG-TCGTCGA (Ij) TCGTCGA-HEG-T-HEG-T (Ik)

It will be immediately apparent that the genus of CICs comprising a corestructure of formula I encompasses CICs comprising a core structure offormula II or III.

In some embodiments, one or more spacers comprises smaller units (e.g.,oligoethylene glycols [e.g., HO—(CH2CH2-O)_(N)—H, where N=2-10, such asHEG and TEG], glycerol, C3 alkyl, and the like) linked together. In oneembodiment, the linkage is an ester linkage (e.g., phosphodiester orphosphorothioate ester) or other linkage, e.g., as described infra.

In certain embodiments, the terminal structures of the CIC arecovalently joined (e.g., nucleic acid moiety-to-nucleic acid moiety;spacer moiety-to-spacer moiety, or nucleic acid moiety-to-spacermoiety), resulting in a circular conformation.

B. Branched CICs

In one embodiment, the CIC comprises the core structure[N_(v)]_(A)---S_(p)  (IV)where S_(p) is a multivalent spacer covalently bonded to the quantity“A” independently selected nucleic acid moieties N_(v), and where A isat least 3, e.g., exactly 3, 4, 5, 6, or 7 or more than 7. In variousembodiments, A is L integer between 3 and 100 (inclusive). In someembodiments, A is an integer in a range defined by a lower limit ofabout 3, 5, 10, 50, or 100 and an independently selected upper limit ofabout 5, 7, 10, 50, 100, 150, 200, 250, or 500. It is also contemplatedthat in some embodiments, A is greater than about 500.

In a related embodiment, the CIC comprises the core structure[S_(v)—N_(v)]_(A)---S_(p)  (V)where S_(p) is a multivalent spacer covalently bonded to the quantity“A” independently selected elements, S_(v)—N_(v), comprising a spacermoiety covalently bound to a nucleic acid moiety, and where A is atleast 3. In various embodiments, A is an integer between 3 and 100(inclusive). In some embodiments, A is an integer in a range defined bya lower limit of 5, 10, 50, or 100 and an independently selected upperlimit of 10, 50, 100, 250, or 500. It is also contemplated that in someembodiments, A is greater than 500. In a related embodiment, the CICcomprises the core structure:(S₁—N₁)—S_(p)--(N_(v))_(A)  (VI)where S_(p) is a multivalent spacer covalently bonded to the quantity“A” independently selected nucleic acid moieties, N_(v), and at leastone nucleic acid moiety N₁ bound to a spacer moiety S₁, where A is atleast 2. In one embodiment, A is 2. In various embodiments, A is 3, is4, is 5, or is an integer between 3 and 100 (inclusive). In someembodiments, A is an integer in a range defined by a lower limit of 5,10, 50, or 100 and an independently selected upper limit of 10, 50, 100,150, 200, 250, or 500. It is also contemplated that in some embodiments,A is greater than 500. In some embodiments of the invention, the CIC hasthe structure of formula I, II or III. However, according to theinvention, branched CICs comprise, but are not limited to, thestructures provided in formulas IV, V and VI. That is, formulas IV, Vand VI define core structures in which a multivalent spacer moiety(S_(p)) is covalently bound to at least three (3) nucleic acid moieties.It is contemplated that, in some embodiments, additional chemicalmoieties (e.g., phosphate, mononucleotide, additional nucleic acidand/or spacer moieties) are covalently bound at the termini of the corestructures. For example, if all nucleic acid moieties in a CIC are5′TCGTCGA 3′ and all spacer moieties are glycerol or HEG, CICs having acore structure of formula IV include:

(TCGTCGA)₂-glycerol-TCGTCGA (IVa) (TCGTCGA-HEG)₂-glycerol-TCGTCGA (IVb)(TCGTCGA-HEG-TCGTCGA)₂-glycerol-TCGTCGA (IVc)[(TCGTCGA)₂-glycerol-TCGTCGA]₂-glycerol-TCGTCGA (IVd)

It will be immediately apparent, for example, that the genus of CICscomprising a core structure of formula IV encompasses CICs comprising acore structure of formula V or VI. In a preferred embodiment of theinvention, the CIC comprises at least two different (i.e., differentsequence) nucleic acid moieties.

In some embodiments, one or more spacers comprises smaller units (e.g.,HEG, TEG, glycerol, C3 alkyl, and the like) linked together. In oneembodiment, the linkage is an ester linkage (e.g., phosphodiester orphosphorothioate ester).

A non-nucleic acid spacer moiety that is covalently linked to more thantwo nucleic acid moieties can be referred to as a “multivalent spacer.”As is discussed below, examples of multivalent spacers include glycerol,FICOLL®, and dendrimer moieties that are covalently linked to more thantwo nucleic acid moieties. (Glycerol, for example, can also be a linearspacer, if it is linked to only two nucleic acid moieties; see Example11.)

For convenience, a multivalent spacer with a low valency is sometimescalled a “branched spacer” or “branching spacer.” A multivalent spacerwith low valency is a multivalent spacer that is readily covalentlylinked to not more than 10 nucleic acid moieties, usually fewer than 6,sometimes fewer than 4 and sometimes 3 nucleic acid moieties often or,)Examples of multivalent spacer with a low valency include glycerol,1,3-diamino-2-propanol and substituted derivatives (e.g., “symmetricaldoubler”), pentaerythritol deriviatives (e.g., “trebler”), and the like.In contrast, multivalent spacers can readily covalently bind >10 nucleicacid moieties, and are often are capable of covalent linkageto >50, >100 or >200 nucleic acid moieties. Examples of multivalentspacer with a high valency include Ficoll®, dextran, and other modifiedpolysaccharides, “Starburst® dendrimers of Generation 2-5 (valency16-128), and the like.

C. CICs Having Specified Tertiary Structure, and CIC Multimers

The linear and branched CICs described herein (e.g., in Sections B andC, supra) include variants having particular structural features. CICsand CIC multimers described in this section may be targeted to, orefficiently taken up by phagocytic cells or antigen-presenting cells,may present a high density of nucleic acid moiety 5′-ends, may changestructure in vivo (e.g., due to nuclease or other degradative activity,acidification in the endosome, and/or dilution of the CIC or multimer invivo (thereby changing properties after administration to a subject orin a particular biological compartment).

i) CICs Having Specified Tertiary Structure

As noted elsewhere herein, linear CICs with at least two nucleic acidmoieties having sequences complementary or partially complementary toeach other can form hairpin duplexes (and/or CIC dimers or concatamers,as discussed below). As used herein, “hairpin duplex” refers to thestructure formed by hybridization of two nucleic acid moieties that arein the same orientation in the CIC (e.g., one nucleic acid moiety isbound at the 3′ terminus to the spacer moiety and the other nucleic acidmoiety is bound at the 5′ terminus to the spacer moiety) in a CIC. Inone embodiment, the two nucleic acid moieties are separated by no morethan one additional nucleic acid moiety. In another embodiment, there isno intervening nucleic acid moiety between the base-paired nucleic acidmoieties. Examples of CICs that may form hairpin duplexes, provided forillustration and not limitation, are C-159 and C-160 shown infra inTable 2 and the Examples). Also see FIG. 8A. In a hairpin duplex, thepair of nucleic acid moieties with complementary sequences can beself-complementary (e.g., palindromic) or the pair can have differentsequences. It will be appreciated that exact complementarity is notrequired so long as the nucleic acid moieties are of sufficientcomplementarity and length to form a duplex at 37° C. in an aqueoussolution at physiological pH (i.e., 7.0-7.4, e.g., 7.2) and ionicstrength (e.g., 150 mM NaCl).

The presence of a duplex structure can be detected using well-knownmethods. These include detecting a change in CIC structure based on sizeexclusion chromatography, and detecting a change in A₂₆₀ or A₂₈₀ uponraising or lowering the temperature of the CIC-containing composition(indicative of melting or formation of the duplex). Absorbance increasesas a double-stranded DNA separates into the single-stranded forms.

As noted, certain CICs can form hairpin structures or can form dimers orconcatamers. It is believed the latter structures are favored when theCICs are allowed to anneal at high concentration and/or when the spaceris of sufficient length and flexibility (e.g., [HEG]₆) to favor thekinetics of dimer formation by providing increased degrees of freedom ofmovement of the nucleic acid moieties.

Like linear CICs, branched CICs can form a variety of types ofstructures, including the “fork,” “H,” “comb,” “central spacer,” and“dendrimer” structures described below and in the Examples.

A “fork” structure has only a single branching spacer (e.g. glycerol,glycerol-[HEG]₂, symmetrical doubler-[HEF]₂, and the like), which isbound to three nucleic acid moieties, as illustrated in FIG. 8B (CICC-155; C-35). The three nucleic acid moieties can all have the samesequence, or can have different sequences. In one embodiment, at least 2of the nucleic acid moieties has the same sequence. In one embodiment,at least 1, at least 2, or at least 3 of the nucleic acid moieties is a5-prime moiety (see §3(C), infra for an explanation of thisnomenclature). In an embodiment, at least 1, at least 2, or at least 3of the nucleic acid moieties includes the sequence CG, optionally TCG,optionally 5′^(F)-TCG (i.e., TCG in the 5-prime position of a 5-primemoiety; see §3(C), infra for an explanation of this nomenclature). Thereader will recognize that one or more of the nucleic acid moieties canhave a sequence, motif or property described herein below (e.g.,§III(2)-(3)).

A “trident” structure has only a single branching spacer (e.g., trebler,[HEG]-trebler-[HEG]₃, and the like), which is bound to four nucleic acidmoieties. The four nucleic acid moieties can all have the same sequence,or can have different sequences. In one embodiment, at least 3 of thenucleic acid moieties has the same sequence. In one embodiment, at least1, at least 2, at least 3, or at least 4 of the nucleic acid moieties isa 5-prime moiety. In an embodiment, at least 1, at least 2, at least 3,or at least 4 of the nucleic acid moieties includes the sequence CG,optionally TCG, optionally 5′^(F)-TCG. The reader will recognize thatone or more of the nucleic acid moieties can have a sequence, motif orproperty described herein below (e.g., §III(2)-(3)).

A “polydent” structure has at least 3 branched spacers (e.g., 3-15,usually 3-7) and at least 4 nucleic acid moieties, where all of thenucleic acid moieties in the structure have an unbound terminus (a free5′ end or a free 3′ end). In one embodiment all of the nucleic acidmoieties have a free 5′-end. See, e.g., C-126 (Table 2).

An “H” structure is defined by having exactly two branching spacers,each of which is linked to the other via (a) a nucleic acid moiety or(b) a combination of nucleic acid moieties and nonbranching spacers(e.g., -ATTT-HEG-ATTT-) and each of which is linked to two additionalnucleic acid moieties. An example is CIC C-171 (see FIG. 8C). Inembodiments, at least 1, at least 2, at least 3 or at least 4 (i.e.,all) of the “two additional nucleic acid moieties” is a 5-prime moiety.In one embodiment, at least 1, at least 2, at least 3, or at least 4 ofthe two additional nucleic acid moieties is a 5-prime moiety. Inembodiments, at least 1, at least 2, at least 3, or at least 4 of thenucleic acid moieties includes the sequence CG, optionally TCG,optionally 5′^(F)-TCG. The reader will recognize that one or more of thenucleic acid moieties can have a sequence, motif or property describedherein below (e.g., §III(2)-(3)). The nucleic acid moiety(s) linking thetwo branching spacers may also comprise a sequence CG or other sequenceor motif described herein.

A “comb” structure comprises the following structure VII:

wherein y and z are independently 0 or 1, and n can be from 1 to 10,preferably 3 to 6, most preferably 3 or 4. In this formula, each S(including S₁ and S₂) represents a spacer (which may be the same ordifferent), and is always branched spacer, unless x or y is 0, in whichcase the end spacer(s) [represented by S₁ and S₂] will be linear. N′,N_(y) and N_(Z) are nucleic acid moieties where each N′ has the samesequence. Each X represents the structure [(N-LS)_(m)—N], where m isfrom 0 to 5, usually 0 or 1; where each N (as well as N_(y) and N_(z))is independently selected and represents a nucleic acid moiety which maybe the same or different; and where each LS represents a linear spacer,where each linear spacer is independently selected and may be the sameor different. In various embodiments, at least one nucleic acid moietyis a 5-prime moiety and/or includes the sequence CG, optionally TCG,optionally 5′^(F)-TCG. In an embodiment, each N′ is a 5-prime moiety. Inone embodiment all of the 5-prime moieties have the same sequence and/orall of the nucleic acid moieties that are not 5′ moieties have the samesequence. The reader will recognize that one or more of the nucleic acidmoieties can have a sequence, motif or property described hereinbelow(e.g., §III(2)-(3)). An example of a comb structure is C-169 (see FIG.8D). In comb structures, the branched spacers may be the same.Alternatively, a comb structure may contain 2 or more different branchedspacers.

A “central spacer” structure is defined by having spacer moiety bound to4 or more nucleic acid moieties, where at least 3 of said 4 or morenucleic acid moieties is a 5-prime moiety, and wherein at least 3 of the5-prime moieties include the sequence CG, optionally TCG, optionally5′^(F)-TCG. The reader will recognize that one or more of the nucleicacid moieties can have a sequence, motif or property describedhereinbelow (e.g., §III(2)-(3)). See C-139, C-140, C-168, C-170, andFIGS. 8E, 8F, and 8G. In various embodiments, the number of nucleic acidmoieties bound to the spacer may be less than 500 (e.g., for CICs madeby conjugation strategies, such as CICs with Ficoll-based centralspacers) or less than about 10 (e.g., for compounds made using a DNAsynthesizer, e.g. C-168 and C-170).

A “CIC dendrimer” is a discrete, highly branched polymer created bycovalent linking of multiple (e.g., 3-15) branched CICs. Usually all ormost of the component CICs has the same structural motif (e.g., all arefork structures or all are trident structures). For example, FIG. 8Hshows a third generation CIC dendrimer produced by linking 7 forkstructure CICs. Also see structure IVd in Section §III(1)(b), an exampleof a 2^(nd) generation dendrimer containing 3 fork CICs. The CICdendrimer should not be confused with dendrimers that may serve asspacer moieties but which do not comprise nucleic acid moieties (e.g.,the “dense star or “starburst” polymers described hereinbelow).

ii) CIC Multimers

Certain CIC linear or branched CICs of the invention can form“multimers” of 2 or more CICs that stably associate with each other dueto Watson-Crick hybridization between pairs of at least partiallycomplementary nucleic acid moieties. Examples of such CIC multimers aremultimers comprising only linear CICs (e.g., see FIGS. 9A-D) and CICmultimers comprising at least one, and usually at least two, branchedCICs (e.g., see FIGS. 9E-G).

Examples of CIC multimers comprising only linear CICs include dimersshown in FIG. 9A (showing dimers where the component linear CICs are thesame), FIG. 9B (showing dimers where the component linear CICs aredifferent), FIG. 9C (showing dimers having 5′ ends that are notbasepaired), and FIG. 9D (showing a concatamer of linear CICs). Examplesof CIC multimers comprising branched CICs are shown in FIG. 9E (showinga “central axis” structure), FIG. 9F (showing a “cage” structure), andFIG. 9G (showing a “starfish” structure). It will be understood themultimers of FIG. 9 are provided for illustration and not limitation.Thus, the majority of CIC multimers shown in FIG. 9 are assemblies oftwo CICs. In various alternative embodiments CIC multimers may compriseat least 2, at least 3, at least 4, at least 5, at least 10, andsometimes more than 10 individual CICs. The individual CIC subunits neednot all be the same.

As noted, individual CICs in CIC multimers stably associate with eachother. As used in this context, “stably associate” means the CICs remainassociated at 37° C. in a buffered aqueous salt solution of nearphysiological ionic strength and pH, e.g., 150 mM NaCl, pH 7.2. It willbe recognized, of course, that even “stably associated” multimericmacromolecules may exist in a state of equilibrium such that anindividual CICs may be unassociated with the multimer for relativelybrief periods of time, or there may be exchange between CICs in themultimeric structure and unassociated monomers in solution. CICmultimers may be self assembling (i.e., the component CICs mayspontaneously associate under physiological conditions). Usually, a CICmultimer will form when the component CICs are dissolved at aconcentration of approximately 1.0 mg/ml in 50 mM sodium phosphate/150mM sodium chloride/pH 7.2, heated to 95° C. for 3 min., and allowed toslowly (e.g., over a period of approximately 2 hours) to 37° C. or roomtemperature. See Example 51, infra.

Because the association between CICs in a CIC multimer relies, at leastin part, on hybrids formed between nucleic acid moieties that are atleast partially complementary, and sometimes exactly complementary, thenormal parameters for formation of nucleic acid hybrids apply. That is,the hybridizing regions of nucleic acid moieties are of sufficientlength and/or sequence composition (e.g., GC content) to form stable CICmultimers. Generally the nucleic acid moieties of one CIC will compriseat least 8, more often at least 10, and usually at least 12 contiguousbases that are exactly complementary to nucleic acid moieties of asecond CIC in the multimer. However, when there are a large number ofhybridizing nucleic acid moieties, the region of complementarity orcontiguity may be shorter.

Conditions under which two polynucleotides, or regions of aself-complementary polynucleotide, will form a duplex can be determinedempirically or can be predicted using are well known methods (takinginto consideration base sequence, polynucleotide length, type of esterlinkage [e.g., phosphorothioate or phosphodiester linkage], temperature,ionic strength, presence of modified bases or sugars, etc.). Theannealing nucleic acid moieties in the associating CICs may beselfcomplementary (see, e.g., FIG. 9F) or alternatively, a nucleic acidmoiety(s) on one CIC may be complementary to a nucleic acid moiety(s) ona second CIC, but not to itself.

As noted above, examples of CIC multimers include multimers having a“central axis” structure, a “cage” structure, and a “starfish”structure.” A “central axis” structure refers to a dimer of two branchedCICs, in which one nucleic acid moiety of each CIC forms adouble-stranded region with a complementary nucleic acid moiety of thesecond CIC, and each spacer is bound to at least two other nucleic acidmoieties. See FIG. 9E.

A “cage” structure refers to a CIC multimer in which at least twonucleic acid 5-prime moieties from each component CIC are hybridized toa nucleic acid moiety of another CIC in the multimer. See FIG. 9F. Insome embodiments, all of the 5-prime moieties from one or all of theCICs are hybridized to a nucleic acid moiety of another CIC in themultimer. A “cage” structure is characterized in that each of thenucleic acid 5-prime moieties in a duplex is linked to the spacer moietywith the same polarity (i.e., the spacer moiety-nucleic acid moietylinkage for each nucleic acid moiety in a particular duplex is either 3′or is 5′). In an embodiment, the cage structure CIC multimer contains nomore than two CICs.

A “starfish” structure has the same properties as the cage structure,supra, except (a) the starfish is always a dimer and (b) the two nucleicacid moieties in each duplex are linked to the spacer moieties withdifferent polarities (i.e., one is linked at the 5′ terminus and one islinked at the 3′ terminus). See FIG. 9G.

In each type of CIC multimer, it will be understood that nucleic acidmoieties in the multimer may have any of the sequence, structuralfeatures or properties described herein for nucleic acid moieties, solong as the feature is consistent with the multimer structure. Thus, oneor more nucleic acid moieties may be a 5-prime moiety, may include thesequence CG, TCG, or 5′^(F)-TCG, or have other sequence, motif orproperty described herein (e.g., §III(2)-(3)). Further, it will beunderstood the multimers of FIG. 9 are provided for illustration and notlimitation.

Examples 58 and 59 illustrate that tertiary structure andmultimerization can enhance the activity of CICs. The results of Example58 show that CICs that can self-hybridize (C-173, C-174, C-175) inducedsignificantly more IFN-α from human PBMC than did the parentoligonucleotide (P-17) when used at low doses (e.g., 0.8 ug/ml). Theresults of Example 59 show that CICs that hybridize to produce a totalof four free 5′-ends with active TCG-containing heptameters (e.g.,C—C178 duplex, C-202/C-203 heteroduplex) induced significantly moreIFN-α from human PBMC than CICs containing only two free 5′-ends (C-101,C-202, C-203).

D. Single-Spacer CICs

In a different aspect of the invention, the CIC comprises a structure inwhich there is a single nucleic acid moiety covalently conjugated to asingle spacer moiety, i.e.,N₁—S₁  (VIII)

In one embodiment, S₁ has the structure of a multimer comprising smallerunits (e.g., oligoethylene glycols, [e.g., HO—(CH2CH2-O)_(N)—H, whereN=2-10; e.g., HEG and TEG], glycerol, 1′2′-dideoxyribose, C2 alkyl-C12alkyl subunits [preferably, C2 alkyl-C10 alkyl subunits], and the like),typically connected by an ester linkage (e.g., phosphodiester orphosphorothioate ester), e.g., as described infra. See, e.g., formulaVIIIa, infra. The multimer can be heteromeric or homomeric. In oneembodiment, the spacer is a heteromer of monomeric units (e.g., HEG,TEG, glycerol, 1′2′-dideoxyribose, C2 alkyl to C12 alkyl linkers,preferably C2 alkyl to C10 alkyl linkers, and the like) linked by anester linkage (e.g., phosphodiester or phosphorothioate ester). See,e.g., formula VIIb, infra.

For example, if the nucleic acid moiety is 5′TCGTCGA 3′ and the spacermoiety is a phosphorothioate-linked multimer of hexaethylene glycol[“(HEG)₁₅”], a CIC having a core structure of formula VII includes:

TCGTCGA-(HEG)₁₅ (VIIa)

Similarly, if the nucleic acid moiety is 5′TCGTCGA 3′ and the spacermoiety is a phosphorothioate-linked multimer of alternating hexaethyleneglycol and propyl subunits, a CIC having a core structure of formula VIincludes:

TCGTCGA-HEG-propyl-HEG-propyl-HEG (VIIb).

2. Immunomodulatory Activity of CICs

The CICs of the invention have immunomodulatory activity. The terms“immunomodulatory,” “immunomodulatory activity,” or “modulating animmune response,” as used herein, include immunostimulatory as well asimmunosuppressive effects. An immune response that is immunomodulatedaccording to the present invention is generally one that is shiftedtowards a “Th1-type” immune response, as opposed to a “Th2-type” immuneresponse. Th1-type responses are typically considered cellular immunesystem (e.g., cytotoxic lymphocytes) responses, while Th2-type responsesare generally “humoral”, or antibody-based. Th1-type immune responsesare normally characterized by “delayed-type hypersensitivity” reactionsto an antigen. Th1-type responses can be detected at the biochemicallevel by increased levels of Th1-associated cytokines such as IFN-γ,IFN-α, IL-2, IL-12, and TNF-α, as well as IL-6, although IL-6 may alsobe associated with Th2-type responses as well. Th2-type immune responsesare generally associated with higher levels of antibody production,including IgE production, an absence of or minimal CTL production, aswell as expression of Th2-associated cytokines such as IL-4 and IL-5.

Immunomodulation in accordance with the invention may be recognized bymeasurements (assays) in vitro, in vivo and/or ex vivo. Examples ofmeasurable immune responses indicative of immunomodulatory activityinclude, but are not limited to, antigen-specific antibody production,secretion of cytokines, activation or expansion of lymphocytepopulations such as NK cells, CD4+ T lymphocytes, CD8+ T lymphocytes, Blymphocytes, and the like. See, e.g., WO 97/28259; WO 98/16247; WO99/11275; Krieg et al. (1995) Nature 374:546-549; Yamamoto et al. (1992)J. Immunol. 148:4072-4076; Ballas et al. (1996) J. Immunol.157:1840-1845; Klinman et al. (1997) J. Immunol. 158:3635-3639; Sato etal. (1996) Science 273:352-354; Pisetsky (1996) J. Immunol. 156:421-423;Shimada et al. (1986) Jpn. J. Cancer Res. 77:808-816; Cowdery et al.(1996) J. Immunol. 156:4570-4575; Roman et al. (1997) Nat. Med.3:849-54; Lipford et al. (1997) Eur. J. Immunol. 27:2340-2344; WO98/55495, WO 00/61151, Pichyangkul et al. (2001) J. Imm. Methods247:83-94. See also the Examples, infra. Certain useful assays aredescribed herein below for purposes of illustration and not forlimitation.

Assays are generally carried out by administering or contacting a cell,tissue, animal or the like with a test sample (e.g., containing a CIC,polynucleotide, and/or other agent) and measuring a response. The testsamples containing CICs or polynucleotides can be in a variety of formsor concentrations, which will be understood by the ordinarily skilledpractitioner to be appropriate for the assay type. For example, forpurposes of a cell-based assay, CICs or polynucleotides are often usedat a concentration of 20 μg/ml or 10 μg/ml or 2 μg/ml. Typically, forthe purposes of the assay, concentration is determined by measuringabsorbance at 260 nm and using the conversion 0.5 OD₂₆₀/ml=20 μg/ml.This normalizes the amount of total nucleic acid in the test sample andmay be used, for example, when the spacer moiety does not have asignificant absorbance at 260 nm. Alternatively, concentration or weightcan be measured by other methods known in the art. If desired, theamount of nucleic acid moiety can be determined by measuring absorbanceat 260 nm, and the weight of the CIC calculated using the molecularformula of the CIC. This method is sometimes used when the ratio ofweight contributed by the spacer moiety(s) to weight contributed by thenucleic acid moieties in a CIC is high (i.e., greater than 1).

It will similarly be understood that positive and negative controls areuseful in assays for immunomodulatory activity. A suitable positivecontrol for immunomodulatory activity is the immunomodulatoryphosphorothioate DNA having the sequence 5′-TGACTGTGAACGTTCGAGATGA-3′(SEQ ID NO:2), although other suitable positive controls withimmunomodulatory activity will be apparent to the ordinarily skilledpractitioner. One suitable negative control is no test agent (i.e.,excipient or media alone, also referred to as “cells alone” for certainin vitro assays). Alternatively, a phosphorothioate DNA having thesequence 5′-TGACTGTGAACCTTAGAGATGA-3′ (SEQ ID NO:3) is used as anegative control in some embodiments. Other negative controls can bedesigned by the practitioner guided by the disclosure herein andordinary assay design.

One useful class of assays is “cytokine response assays.” An exemplaryassay for immunomodulatory activity measures the cytokine response ofhuman peripheral blood mononuclear cells (“PBMCs”) (e.g., as describedin Bohle et al. [1999], Eur. J. Immunol. 29:2344-53; Verthelyi et al.[2001] J. Immunol. 166:2372-77). In one embodiment of this assay,peripheral blood is collected from one or more healthy human volunteersand PBMCs are isolated. Typically blood is collected by venipunctureusing a heparinized syringe, layered onto a FICOLL® (Amersham PharmaciaBiotech) cushion and centrifuged. PBMCs are then collected from theFICOLL® interface and washed twice with cold phosphate buffered saline(PBS). The cells are resuspended and cultured (e.g., in 48- or 96-wellplates) at 2×10⁶ cells/mL in RPMI 1640 with 10% heat-inactivated humanAB serum, 50 units/mL penicillin, 50 μg/mL streptomycin, 300 μg/mLglutamine, 1 mM sodium pyruvate, and 1×MEM non-essential amino acids(NEAA) in the presence and absence of test samples or controls for 24hours.

Cell-free medium is collected from each well and assayed for IFN-γand/or IFN-α concentration. Immunomodulatory activity is detected whenthe amount of IFN-γ secreted by PBMCs contacted with the test compoundis significantly greater (e.g., at least about 3-fold greater, usuallyat least about 5-fold greater) than the amount secreted by the PBMCs inthe absence of the test compound or, in some embodiments, in thepresence of an inactive control compound (e.g.,5′-TGACTGTGAACCTTAGAGATGA-3′ (SEQ ID NO:3)). Conversely, a test compounddoes not have immunomodulatory activity if the amount of IFN-γ secretedby PBMCs contacted with the test compound is not significantly greater(e.g., less than 2-fold greater) than in the absence of the testcompound or, alternatively, in the presence of an inactive controlcompound (e.g., 5′-TGACTGTGAACCTTAGAGATGA-3′ (SEQ ID NO:3)).

When IFN-α concentration is assayed, the amount of IFN-α secreted byPBMCs contacted with the test compound is often significantly greater(e.g., in the case of IFN-α sometimes at least about 2-fold or at leastabout 3-fold greater) than the amount secreted by the PBMCs in theabsence of the test compound or, in some embodiments, in the presence ofan inactive control compound (e.g., 5′-TGACTGTGAACCTTAGAGATGA-3′ (SEQ IDNO:3)). In some embodiments, the significantly increased IFN-α secretionlevel is at least about 5-fold, at least about 10-fold, or even at leastabout 20-fold greater than controls. Conversely, a test compound doesnot have immunomodulatory activity if the amount of IFN-α secreted byPBMCs contacted with the test compound is not significantly greater(e.g., less than 2-fold greater) than in the absence of the testcompound or, alternatively, in the presence of an inactive controlcompound (e.g., 5′-TGACTGTGAACCTTAGAGATGA-3′ (SEQ ID NO:3)).

As illustrated in the examples, infra, administration of some CICsresults in significant secretion of both IFN-γ and IFN-α, whileadministration of other CICs has a lesser effect on secretion of IFN-αor, conversely, a lesser effect on secretion of IFN-γ. See, e.g.,Example 49.

Another useful class of assays are cell proliferation assays, e.g., Bcell proliferation assays. The effect of an agent (e.g. a CIC) on B cellproliferation can be determined using any of a variety of assays knownin the art. An exemplary B cell proliferation assay is provided inExample 41.

To account for donor variation, e.g., in cell-based assays, such ascytokine and proliferation assays, preferably assays are carried outusing cells (e.g., PBMCs) from multiple different donors. The number ofdonors is usually at least 2 (e.g. 2), preferably at least 4 (e.g. 4),sometimes at least 10 (e.g. 10). Immunomodulatory activity is detectedwhen the amount of IFN-γ secreted in the presence of the test compound(e.g. in at least half of the healthy donors tested, preferably in atleast 75%, most preferably in at least 85%) is at least about 3-foldgreater or at least about 5-fold greater than secreted in the absence ofthe test compound, or in some embodiments, than in the presence of aninactive control compound such as described supra.

Immunomodulatory activity may also be detected by measuringinterferon-induced changes in expression of cytokines, chemokines andother genes in mammalian cells (e.g., PBMCs, bronchial alveolar lavage(BAL) cells, and other cells responsive to interferon). For example,expression of the chemokines interferon-induced-protein 10 kDa (IP-10),monokine induced by IFN-γ (MIG) and monocyte chemotactic protein 1(MCP-1) are increased in the presence of IFN-α and IFN-γ. Expression ofthese proteins, or their corresponding mRNA, may be used as markers ofimmunostimulatory activity in cultured cells or tissues or blood ofanimals to which a CIC has been administered. Expression of such markerscan be monitored any of a variety of methods of assessing geneexpression, including measurement of mRNAs (e.g., by quantitative PCR),immunoassay (e.g., ELISA), and the like.

Biological activity of CICs can also be measured by measuring theinduction of gene products known to have antiviral activities, including2′-5′ Oligoadenylate synthetase (2′-5′OAS), Interferon-stimulatedgene—54 kD (ISG-54 kD), Guanylate binding protein-1 (GBP-1), M×A andM×B. Expression of these proteins, or their corresponding mRNA, may beused as markers of immunostimulatory activity in cultured cells ortissues or blood of animals to which a CIC has been administered.Expression of such markers can be monitored any of a variety of methodsof assessing gene expression, including measurement of mRNAs (e.g., byquantitative PCR), immunoassay (e.g., ELISA), and the like.

In vitro assays can also be carried out using mouse cells, as described,for example, in Example 42, infra, and in other mammalian cells.

Exemplary in vivo assays are described in Examples 43, 44, and 46 (mice)and Example 45 (non-human primates).

Except where otherwise indicated or apparent, the cytokine assaysdescribed in the Examples, infra, are conducted using human PBMCs usingessentially the protocol described in Example 28. Large numbers of testcompounds can be assayed simultaneously, e.g., using multi-well platesor other multi-chamber assay materials. If desired, the assays can becarried out by computer-controlled robotic mechanisms well known in theart.

3. Nucleic Acid Moieties

The CICs of the invention comprise one or more nucleic acid moieties.The term “nucleic acid moiety,” as used herein, refers to a nucleotidemonomer (i.e., a mononucleotide) or polymer (i.e., comprising at least 2contiguous nucleotides). As used herein, a nucleotide comprises (1) apurine or pyrimidine base linked to a sugar that is in an ester linkageto a phosphate group, or (2) an analog in which the base and/or sugarand/or phosphate ester are replaced by analogs, e.g., as describedinfra. In a CIC comprising more than one nucleic acid moiety, thenucleic acid moieties may be the same or different.

The next three sections describe characteristics of nucleic acidmoieties such as length, the presence, and the position of sequences orsequence motifs in the moiety, as well as describing (without intendingto limit the invention) the properties and structure of nucleic acidmoieties and CICs containing the moieties.

A. Length

Usually, a nucleic acid moiety is from 1 to 100 nucleotides in length,although longer moieties are possible in some embodiments. In someembodiments, the length of one or more of the nucleic acid moieties in aCIC is less than 8 nucleotides (i.e., 1, 2, 3, 4, 5, 6 or 7nucleotides). In various embodiments, a nucleic acid moiety (such as anucleic acid moiety fewer than 8 nucleotides in length) is at least 2nucleotides in length, often at least 3, at least 4, at least 5, atleast 6, or at least 7 nucleotides in length. In other embodiments, thenucleic acid moiety is at least 10, at least 20, or at least 30nucleotides in length.

As shown in the Examples infra, CICs containing only heptameric,hexameric, pentameric, tetrameric, and trimeric nucleic acid moietieswere active in assays for immunostimulatory activity (e.g., Examples 36and 37). Thus, it is contemplated that, in some embodiments, a CIC willcomprise at least one nucleic acid moiety shorter than 8 nucleotides. Insome embodiments, all of the nucleic acid moieties in a CIC will beshorter than 8 nucleotides (e.g., having a length in a range defined bya lower limit of 2, 3, 4, 5, of 6 and an independently selected upperlimit of 5, 6, or 7 nucleotides, where the upper limit is higher thanthe lower limit). For example, in one embodiment, specified nucleic acidmoieties in a CIC (including all of the nucleic acid moieties in theCIC) may be either 6 or 7 nucleotides in length. In one embodiment, theCIC comprises two spacer moieties and an intervening nucleic acid moietythat is less than 8 bases in length (e.g., 5, 6, or 7 bases in length).

It is contemplated that in a CIC comprising multiple nucleic acidmoieties, the nucleic acid moieties can be the same or differentlengths. In one embodiment, the length of one or more, or most (e.g., atleast about 2, at least about 4, or at least about 25%, at least about50%, at least about 75%) or all of the nucleic acid moieties in a CIC isfewer than 8 nucleotides, in some embodiments fewer than 7 nucleotides,in some embodiments fewer than 6 nucleotides, in some embodimentsbetween 2 and 6 nucleotides, in some embodiments between 2 and 7nucleotides, in some embodiments between 3 and 7 nucleotides, in someembodiments between 4 and 7 nucleotides, in some embodiments between 5and 7 nucleotides, and in some embodiments between 6 and 7 nucleotides.

As is discussed in greater detail infra, often at least one nucleic acidmoiety of a CIC includes the sequence CG, e.g. TCG, or a CG-containingmotif described herein. In one embodiment, at least one nucleic acidmoiety comprises a CG-containing nucleic acid motif and is less than 8nucleotides in length (e.g., has a specified length as described supraless than 8 nucleotides). In a related embodiment, none of the nucleicacid moieties in a CIC that are longer than 8 nucleotides comprise thesequence “CG” or optionally the sequence “TCG” or “ACG” (i.e., all ofthe nucleic acid moieties in the CIC that comprise the sequence CG areless than 8 nucleotides in length). In an embodiment, at least onenucleic acid moiety in the CIC does not comprise a CG sequence.

B. Sequences and Motifs

As noted supra, a particular nucleic acid moiety can have a variety oflengths. In one embodiment, the nucleic acid moiety has a length shorterthan 8 nucleotides. In one embodiment, the nucleic acid moiety has alength of 8 nucleotides or longer. In various embodiments at least onenucleic acid moiety of a CIC of the invention comprises a sequence asdisclosed infra.

In the formulas provided below, all sequences are in the 5′→3′ directionand the following abbreviations are used: B=5-bromocytosine;bU=5-bromouracil; a-A=2-amino-adenine; g=6-thio-guanine;t=4-thio-thymine. H=a modified cytosine comprising anelectron-withdrawing group, such as halogen in the 5 position. Invarious embodiments, a cytosine (C) in a sequence referred to infra isreplaced with N4-ethylcytosine or N4-methylcytosine or5-hydroxycytisine. In various embodiments, a guanosine (G) in theformula is replaced with 7-deazaguanosine.

In CICs tested thus far, the presence of CG correlates withcytokine-inducing activity. Thus, in one embodiment, at least onenucleic acid moiety of a CIC comprises at least one 5′-cytosine,guanine-3′ (5′-CG-3′) sequence. The cytosine is not methylated at theC-5 position and, preferably is not methylated at any position.

In one embodiment, one or more nucleic acid moieties comprises 3 to 7bases. In one embodiment, the nucleic acid moiety comprises 3 to 7 basesand has the sequence 5′-[(X)₀₋₂]TCG[(X)₂₋₄]-3′, or 5′-TCG[(X)₂₋₄]-3′, or5′-TCG(A/T)[(X)₁₋₃]-3′, or 5′-TCG(A/T)CG(A/T)-3′, or 5′-TCGACGT-3′ or5′-TCGTCGA-3′, wherein each X is an independently selected nucleotide.In some embodiments, the CIC contains at least 3, at least 10, at least30 or at least 100 nucleic acid moieties having an aforementionedsequence.

In an embodiment, the nucleic acid moiety comprises the sequence5′-thymidine, cytosine, guanine-3′ (5′-TCG-3′), for example (withoutlimitation), the 3-mer TCG, the 4-mer TCGX (e.g., TCGA), the 5-mersTCGXX (e.g., TCGTC and TCGAT), the 6-mers TCGXXX, XTCGXX and TCGTCG, andthe 7-mers TCGXXXX, XTCGXXX, XXTCGXX and TCGTCGX, where X is any base.Often, at least one nucleic acid moiety comprises the sequence5′-thymidine, cytosine, guanine, adenosine-3′ (5′-TCGA-3′), e.g.,comprises a sequence 5′-TCGACGT-3′. In one embodiment, the nucleic acidmoiety comprises a heptameric sequence 5′-TCGXCGX, 5′-TCGXTCG,5′-TCGXXCG, 5′-TCGCGXX where X is any base. In some embodiments theaforementioned sequence is located at the 5-prime position of a CIC,e.g., 5′^(F)-TCGXCGX, 5′^(F)-TCGXTCG, 5′^(F)-TCGXXCG, 5′^(F)-TCGCGXX′.CICs comprising these sequences have been discovered to be particularlyeffective for induction of IFN secretion. For example, compare theresults in FIG. 10 for C-41 with C-21, C-74 with C-51, and C-143 withC-94, C-142, and C-158.

In some embodiments, a nucleic acid moiety comprises the sequence5′-ACGTTCG-3′; 5′-TCGTCG-3′; 5′-AACGTTC-3′; 5′-AACGTT-3′; 5′-TCGTT-3′;5′-CGTTCG-3′; 5′-TCGTCGA-3′; 5′-TCGXXX-3′; 5′-XTCGXX-3′; 5′-XXTCGX-3′;5′-TCGAGA-3′; 5′-TCGTTT-3′; 5′-TTCGAG-3′; 5′-TTCGT-3′; 5′-TTCGC-3′;5′-GTCGT-3′; 5′-ATCGT-3′; 5′-ATCGAT-3′; 5′-GTCGTT-3′; 5′-GTCGAC-3′;5′-ACCGGT-3′; 5′-AABGTT-3′; 5′-AABGUT-3′, 5′-TCGTBG-3′ where X is anynucleotide.

In some embodiments, a nucleic acid moiety comprises a sequence that is5′-purine, purine, C, G, pyrimidine, pyrimidine-3′; 5′-purine, purine,C, G, pyrimidine, pyrimidine, C, G-3′; or 5′-purine, purine, C, G,pyrimidine, pyrimidine, C, C-3′; for example (all 5′→3′), GACGCT;GACGTC; GACGTT; GACGCC; GACGCU; GACGUC; GACGUU; GACGUT; GACGTU; AGCGTT;AGCGCT; AGCGTC; AGCGCC; AGCGUU; AGCGCU; AGCGUC; AGCGUT; AGCGTU; AACGTC;AACGCC; AACGTT; AACGCT; AACGUC; AACGUU; AACGCU; AACGUT; AACGTU; GGCGTT;GGCGCT; GGCGTC; GGCGCC; GGCGUU; GGCGCU; GGCGUC; GGCGUT; GGCGTU, AACGTT,AGCGTC, GACGTT, GGCGTT, AACGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC,GGCGCC, AGCGCT, GACGCT, GGCGCT, GGCGTT, and AACGCC. In some embodiments,a nucleic acid moiety comprises the sequence: 5′-purine, purine,cytosine, guanine, pyrimidine, pyrimidine, cytosine, cytosine-3′ or5′-purine, purine, cytosine, guanine, pyrimidine, pyrimidine, cytosine,guanine-3′.

In some embodiments, a nucleic acid moiety comprises a sequence (all5′→3′) AACGTTCG; AACGTTCC; AACGUTCG; AABGTTCG; AABGUTCG and/or AABGTTBG.

In various embodiments, a nucleic acid moiety comprises the motif 5′-X₁X₂ A X₃ C G X₄ T C G-3′ (SEQ ID NO:4) wherein X₁ is T, G, C or B,wherein X₂ is T, G, A or U, wherein X₃ is T, A or C, wherein X₄ is T, Gor U and wherein the sequence is not 5′-TGAACGTTCG-3′ (SEQ ID NO:5) or5′-GGAACGTTCG-3′ (SEQ ID NO:6). Examples include (all 5′→3′): TGAACGUTCG(SEQ ID NO:7); TGACCGTTCG (SEQ ID NO:8); TGATCGGTCG (SEQ ID NO:9);TGATCGTTCG (SEQ ID NO:10); TGAACGGTCG (SEQ ID NO:11); GTAACGTTCG (SEQ IDNO:12); GTATCGGTCG (SEQ ID NO:13); GTACCGTTCG (SEQ ID NO:14); GAACCGTTCG(SEQ ID NO:15); BGACCGTTCG (SEQ ID NO:16); CGAACGTTCG (SEQ ID NO:17);CGACCGTTCG (SEQ ID NO:18); BGAACGTTCG (SEQ ID NO:19); TTAACGUTCG (SEQ IDNO:20); TUAACGUTCG (SEQ ID NO:21) and TTAACGTTCG (SEQ ID NO:22).

In various embodiments, a nucleic acid moiety comprises a sequence:

(SEQ ID NO: 23) 5′-TCGTCGAACGTTCGTTAACGTTCG-3′; (SEQ ID NO: 24)5′-TGACTGTGAACGUTCGAGATGA-3′; (SEQ ID NO: 25)5′-TCGTCGAUCGUTCGTTAACGUTCG-3′; (SEQ ID NO: 26)5′-TCGTCGAUCGTTCGTUAACGUTCG-3′; (SEQ ID NO: 27)5′-TCGTCGUACGUTCGTTAACGUTCG-3′; (SEQ ID NO: 28)5′-TCGTCGAa-ACGUTCGTTAACGUTCG-3′; (SEQ ID NO: 29)5′-TGATCGAACGTTCGTTAACGTTCG-3; (SEQ ID NO: 30)5′-TGACTGTGAACGUTCGGTATGA-3′; (SEQ ID NO: 31)5′-TGACTGTGACCGTTCGGTATGA-3′; (SEQ ID NO: 32)5′-TGACTGTGATCGGTCGGTATGA-3′; (SEQ ID NO: 33) 5′-TCGTCGAACGTTCGTT-3′;(SEQ ID NO: 34) 5′-TCGTCGTGAACGTTCGAGATGA-3′; (SEQ ID NO: 35)5′-TCGTCGGTATCGGTCGGTATGA-3′; (SEQ ID NO: 36) 5′-CTTCGAACGTTCGAGATG-3′;(SEQ ID NO: 37) 5′-CTGTGATCGTTCGAGATG-3′; (SEQ ID NO: 38)5′-TGACTGTGAACGGTCGGTATGA-3′; (SEQ ID NO: 39)5′-TCGTCGGTACCGTTCGGTATGA-3′; (SEQ ID NO: 40)5′-TCGTCGGAACCGTTCGGAATGA-3′; (SEQ ID NO: 41) 5′-TCGTCGAACGTTCGAGATG-3′;(SEQ ID NO: 42) 5′-TCGTCGTAACGTTCGAGATG-3′; (SEQ ID NO: 43)5′-TGACTGTGACCGTTCGGAATGA-3′; (SEQ ID NO: 44)5′-TCGTCGAACGTTCGAACGTTCG-3′; (SEQ ID NO: 45) 5′-TBGTBGAACGTTCGAGATG-3′;(SEQ ID NO: 46) 5′-TCGTBGAACGTTCGAGATG-3′; (SEQ ID NO: 47)5′-TCGTCGACCGTTCGGAATGA-3′; (SEQ ID NO: 48) 5′-TBGTBGACCGTTCGGAATGA-3′;(SEQ ID NO: 49) 5′-TCGTBGACCGTTCGGAATGA-3′; (SEQ ID NO: 50)5′-TTCGAACGTTCGTTAACGTTCG-3′; (SEQ ID NO: 51) 5′-CTTBGAACGTTCGAGATG-3′;(SEQ ID NO: 52) 5′-TGATCGTCGAACGTTCGAGATG-3′.

In some embodiments, a nucleic acid moiety comprises the sequence:5′-X₁X₂ A X₃ B G X₄ T C G-3′ (SEQ ID NO:53), wherein X₁ is T, G, C or B,wherein X₂ is T, G, A or U, wherein X₃ is T, A or C, wherein X₄ is T, Gor U. In some embodiments, the nucleic acid moiety is not5′-TGAABGTTCG-3′ (SEQ ID NO:54). Examples include (all 5′→3′):TGAABGUTCG (SEQ ID NO:55); TGACBGTTCG (SEQ ID NO:56); TGATBGGTCG (SEQ IDNO:57); GTATBGGTCG (SEQ ID NO:58); GTACBGTTCG (SEQ ID NO:59); GAACBGTTCG(SEQ ID NO:60); GAAABGUTCG (SEQ ID NO:61); BGACBGTTCG (SEQ ID NO:62);CGAABGTTCG (SEQ ID NO:63); BGAABGTTCG (SEQ ID NO:64); BGAABGUTCG (SEQ IDNO:65); TTAABGUTCG (SEQ ID NO:66); TUAABGUTCG (SEQ ID NO:67) andTTAABGTTCG (SEQ ID NO:68).

In some embodiments, a nucleic acid moiety comprises the sequence:

(SEQ ID NO: 69) 5′-TGACTGTGAABGUTCGAGATGA-3′; (SEQ ID NO: 70)5′-TCGTCGAABGTTCGTTAABGTTCG-3′; (SEQ ID NO: 71)5′-TGACTGTGAABGUTCGGTATGA-3′; (SEQ ID NO: 72)5′-TGACTGTGAABGUTCGGAATGA-3′; (SEQ ID NO: 73)5′-TCGTCGGAAABGUTCGGAATGA-3′; (SEQ ID NO: 74)5′-TCGTBGAABGUTCGGAATGA-3′.

In some embodiments, a nucleic acid moiety comprises the sequence: 5′-X₁X₂ A X₃ C G X₄ T C G-3′ (SEQ ID NO:75) wherein X₁ is T, C or B, whereinX₂ is T, G, A or U, wherein X₃ is T, A or C, wherein X₄ is T, G or U. Insome embodiments, the formula is not 5′-TGAACGTTCG-3′ (SEQ ID NO:5)

In other embodiments, the nucleic acid moiety comprises the sequence:

(SEQ ID NO: 76) 5′-TGACTGTGAABGTTCGAGATGA-3′; (SEQ ID NO: 77)5′-TGACTGTGAABGTTBGAGATGA-3′; (SEQ ID NO: 78)5′-TGACTGTGAABGTTCCAGATGA-3′; (SEQ ID NO: 79)5′-TGACTGTGAACGTUCGAGATGA-3′; (SEQ ID NO: 80)5′-TGACTGTGAACGbUTCGAGATGA-3′; (SEQ ID NO: 81)5′-TGACTGTGAABGTTCGTUATGA-3′; (SEQ ID NO: 82)5′-TGACTGTGAABGTTCGGTATGA-3′; (SEQ ID NO: 83) 5′-CTGTGAACGTTCGAGATG-3′;(SEQ ID NO: 84) 5′-TBGTBGTGAACGTTCGAGATGA-3′; (SEQ ID NO: 85)5′-TCGTBGTGAACGTTCGAGATGA-3′; (SEQ ID NO: 86)5′-TGACTGTGAACGtTCGAGATGA-3′; (SEQ ID NO: 87)5′-TGACTGTGAACgTTCgAGATGA-3′; (SEQ ID NO: 88)5′-TGACTGTGAACGTTCGTUATGA-3′; (SEQ ID NO: 89)5′-TGACTGTGAACGTTCGTTATGA-3′; (SEQ ID NO: 90)5′-TCGTTCAACGTTCGTTAACGTTCG-3′; (SEQ ID NO: 91)5′-TGATTCAACGTTCGTTAACGTTCG-3′; (SEQ ID NO: 92)5′-CTGTCAACGTTCGAGATG-3′; (SEQ ID NO: 93) 5′-TCGTCGGAACGTTCGAGATG-3′;(SEQ ID NO: 94) 5′-TCGTCGGACGTTCGAGATG-3′; (SEQ ID NO: 95)5′-TCGTCGTACGTTCGAGATG-3′; (SEQ ID NO: 96) 5′-TCGTCGTTTCGTTCGAGATG-3′.

In some embodiments, with respect to any of the sequences disclosedsupra, the nucleic acid moiety further comprises one, two, three or moreTCG and/or TBG and/or THG, sequences, preferably 5′ to the sequenceprovided supra. The TCG(s) and/or TBG(s) may or may not be directlyadjacent to the sequence shown. For example, in some embodiments, anucleic acid moiety includes any of the following: 5′-TCGTGAACGTTCG-3′(SEQ ID NO:97); 5′-TCGTCGAACGTTCG-3′ (SEQ ID NO:98); 5′-TBGTGAACGTTCG-3′(SEQ ID NO:99); 5-TBGTBGAACGTTCG-3′ (SEQ ID NO:100); 5′-TCGTTAACGTTCG-3′(SEQ ID NO:101). In some embodiments, the additional TCG and/or TBGsequence(s) is immediately 5′ and adjacent to the reference sequence. Inother embodiments, there is a one or two base separation.

In some embodiments, a nucleic acid moiety has the sequence:5′-(TCG)_(w) N_(y) A X₃C G X₁ T C G-3′ (SEQ ID NO:102) wherein w is 1-2,wherein y is 0-2, wherein N is any base, wherein X₃ is T, A or C,wherein X₄ is T, G or U.

In some embodiments, the nucleic acid moiety comprises any of thefollowing sequences: TCGAACGTTCG (SEQ ID NO:103); TCGTCGAACGTTCG (SEQ IDNO:98); TCGTGAACGTTCG (SEQ ID NO:97); TCGGTATCGGTCG (SEQ ID NO:106);TCGGTACCGTTCG (SEQ ID NO:107); TCGGAACCGTTCG (SEQ ID NO:108);TCGGAACGTTCG (SEQ ID NO:109); TCGTCGGAACGTTCG (SEQ ID NO:110);TCGTAACGTTCG (SEQ ID NO:111); TCGACCGTTCG (SEQ ID NO:112);TCGTCGACCGTTCG (SEQ ID NO:113); TCGTTAACGTTCG (SEQ ID NO:101).

In some embodiments, a nucleic acid moiety comprises any of thefollowing sequences: 5′-(TBG)_(z) N_(y) A X₃ C G X₄ T C G-3′ (SEQ IDNO:115) wherein z is 1-2, wherein y is 0-2, wherein B is5-bromocytosine, wherein N is any base, wherein X₃ is T, A or C, whereinX₄ is T, G or U.

In some embodiments, a nucleic acid moiety comprises:

(SEQ ID NO: 99) TBGTGAACGTTCG; (SEQ ID NO: 117) TBGTBGTGAACGTTCG;(SEQ ID NO: 118) TBGAACGTTCG; (SEQ ID NO: 100) TBGTBGAACGTTCG;(SEQ ID NO: 119) TBGACCGTTCG; (SEQ ID NO: 120) TBGTBGACCGTTCG.

In some embodiments, a nucleic acid moiety comprises any of thefollowing sequences: 5′-T C G T B G N_(y) A X₃C G X₄ T C G-3′ (SEQ IDNO:121) wherein y is 0-2, wherein B is 5-bromocytosine, wherein N is anybase, wherein X₃ is T, A or C, wherein X₄ is T, G or U. In someembodiments, the nucleic acid moiety comprises any of the followingsequences: TCGTBGTGAACGTTCG (SEQ ID NO:122); TCGTBGAACGTTCG (SEQ IDNO:123); TCGTBGACCGTTCG (SEQ ID NO:124).

In some embodiments, a nucleic acid moiety comprises any of thefollowing sequences: 5′-(TCG)_(w) N_(y) A X₃ B G X₄ T C G-3′ (SEQ IDNO:125) wherein w is 1-2, wherein y is 0-2, wherein N is any base,wherein X₃ is T, A or C, wherein X₄ is T, G or U. In some embodiments,the nucleic acid moiety comprises any of the following sequences:TCGGAAABGTTCG (SEQ ID NO:126) or TCGAABGTTCG (SEQ ID NO:127).

In some embodiments, a nucleic acid moiety comprises any of thefollowing sequences: 5′-(TBG)_(z) N_(y) A X₃ B G X₄ T C G-3′ (SEQ IDNO:128) wherein z is 1-2, wherein y is 0-2, wherein B is5-bromocytosine, wherein N is any base, wherein X₃ is T, A or C, whereinX₄ is T, G or U. In some embodiments, the nucleic acid moiety comprisesany of the following sequences: TBGAABGUTCG (SEQ ID NO:129) orTBGAABGTTCG (SEQ ID NO:130).

In some embodiments, a nucleic acid moiety comprises any of thefollowing sequences: 5′-T C G T B G N_(y) A X₃ B G X₄ T C G-3′ (SEQ IDNO:131) wherein y is 0-2, wherein B is 5-bromocytosine, wherein N is anybase, wherein X₃ is T, A or C, wherein X₄ is T, G or U. In someembodiments, the nucleic acid moiety comprises any of the followingsequences: TCGTBGAABGUTCG (SEQ ID NO:132) or TCGTBGAABGTTCG (SEQ IDNO:133).

In some embodiments, a nucleic acid moiety comprises the sequence:

AACGTTCC, AACGTTCG, GACGTTCC, GACGTTCG.

In some embodiments, a nucleic acid moiety comprises the sequence:

GGCGTTCG; GGCGCTCG; GGCGTCCG; GGCGCCCG; GACGTTCC;GACGCTCC; GACGTCCC; GACGCCCC; AGCGTTCC; AGCGCTCC;AGCGTCCC; AGCGCCCC; AACGTTCC; AACGCTCC; AACGTCCC;AACGCCCC; GGCGTTCC; GGCGCTCC; GGCGTCCC; GGCGCCCC;GACGTTCG; GACGCTCG; GACGTCCG; GACGCCCG; AGCGTTCG;AGCGCTCG; AGCGTCCG; AGCGCCCG; AACGTTCG; AACGCTCG;AACGTCCG; AACGCCCG; GACGCTCC; GACGCCC; AGCGTTCC;AGCGCTCC; AGCGTCCC; AGCGCCCC; AACGTCCC; AACGCCCC;GGCGTTCC; GGCGCTCC; GGCGTCCC; GGCGCCCC; GACGCTCG;GACGTCCG; GACGCCCG; AGCGTTCG; AGCGTCCG; AGCGCCCG; AACGTCCG; AACGCCCG.

In some embodiments, a nucleic acid moiety comprises the sequence:

(5′→3′) TCGTCGA; TCGTCG; TCGTTT; TTCGTT; TTTTCG;ATCGAT; GTCGAC; GTCGTT; TCGCGA; TCGTTTT; TCGTC;TCGTT; TCGT; TCG; ACGTTT; CCGTTT; GCGTTT; AACGTT;TCGAAAA; TCGCCCC; TCGGGGG.

In some embodiments, a nucleic acid moiety comprises an RNA of thesequence AACGUUCC, AACGUUCG, GACGUUCC, and GACGUUCG.

In some embodiments, a nucleic acid moiety has a sequence comprising asequence or sequence motif described in copending coassigned U.S. patentapplication Ser. No. 09/802,685 (published as U.S. ApplicationPublication No. 20020028784A1 on Mar. 7, 2002 and as WO 01/68077 on Sep.20, 2001); Ser. No. 09/802,359 (published as WO 01/68144 on Sep. 20,2001), and copending U.S. application Ser. No. 10/033,243, or in PCTpublications WO 97/28259, WO 98/16247; WO 98/55495; WO 99/11275; WO99/62923; and WO 01/35991. The nucleic acid moiety can also have asequence comprising any or several of the sequences previously reportedto be correlated with immunostimulatory activity when administered as apolynucleotide greater (often substantially greater) than 8 nucleotidesin length, e.g., Kandimalla et al., 2001, Bioorg. Med. Chem. 9:807-13;Krieg et al. (1989) J. Immunol. 143:2448-2451; Tokunaga et al. (1992)Microbiol. Immunol. 36:55-66; Kataoka et al. (1992) Jpn. J. Cancer Res.83:244-247; Yamamoto et al. (1992) J. Immunol. 148:4072-4076; Mojcik etal. (1993) Clin. Immuno. and Immunopathol. 67:130-136; Branda et al.(1993) Biochem. Pharmacol. 45:2037-2043; Pisetsky et al. (1994) LifeSci. 54(2):101-107; Yamamoto et al. (1994a) Antisense Research andDevelopment. 4:119-122; Yamamoto et al. (1994b) Jpn. J. Cancer Res.85:775-779; Raz et al. (1994) Proc. Natl. Acad. Sci. USA 91:9519-9523;Kimura et al. (1994) J. Biochem. (Tokyo) 116:991-994; Krieg et al.(1995) Nature 374:546-549; Pisetsky et al. (1995) Ann. N.Y. Acad. Sci.772:152-163; Pisetsky (1996a) J. Immunol. 156:421-423; Pisetsky (1996b)Immunity 5:303-310; Zhao et al. (1996) Biochem. Pharmacol. 51:173-182;Yi et al. (1996) J. Immunol. 156:558-564; Krieg (1996) Trends Microbiol.4(2):73-76; Krieg et al. (1996) Antisense Nucleic Acid Drug Dev.6:133-139; Klinman et al. (1996) Proc. Natl. Acad. Sci. USA.93:2879-2883; Raz et al. (1996); Sato et al. (1996) Science 273:352-354;Stacey et al. (1996) J. Immunol. 157:2116-2122; Ballas et al. (1996) J.Immunol. 157:1840-1845; Branda et al. (1996) J. Lab. Clin. Med.128:329-338; Sonehara et al. (1996) J. Interferon and Cytokine Res.16:799-803; Klinman et al. (1997) J. Immunol. 158:3635-3639; Sparwasseret al. (1997) Eur. J. Immunol. 27:1671-1679; Roman et al. (1997) NatMed. 3:849-54; Carson et al. (1997) J. Exp. Med. 186:1621-1622; Chace etal. (1997) Clin. Immunol. and Immunopathol. 84:185-193; Chu et al.(1997) J. Exp. Med. 186:1623-1631; Lipford et al. (1997a) Eur. J.Immunol. 27:2340-2344; Lipford et al. (1997b) Eur. J. Immunol.27:3420-3426; Weiner et al. (1997) Proc. Natl. Acad. Sci. USA94:10833-10837; Macfarlane et al. (1997) Immunology 91:586-593; Schwartzet al. (1997) J. Clin. Invest. 100:68-73; Stein et al. (1997) AntisenseTechnology, Ch. 11 pp. 241-264, C. Lichtenstein and W. Nellen, Eds., IRLPress; Wooldridge et al. (1997) Blood 89:2994-2998; Leclerc et al.(1997) Cell. Immunol. 179:97-106; Kline et al. (1997) J. Invest. Med.45(3):282A; Yi et al. (1998a) J. Immunol. 160:1240-1245; Yi et al.(1998b) J. Immunol. 160:4755-4761; Yi et al. (1998c) J. Immunol.160:5898-5906; Yi et al. (1998d) J. Immunol. 161:4493-4497; Krieg (1998)Applied Antisense Oligonucleotide Technology Ch. 24, pp. 431-448, C. A.Stein and A. M. Krieg, Eds., Wiley-Liss, Inc.; Krieg et al. (1998a)Trends Microbiol. 6:23-27; Krieg et al. (1998b) J. Immunol.161:2428-2434; Krieg et al. (1998c) Proc. Natl. Acad. Sci. USA95:12631-12636; Spiegelberg et al. (1998) Allergy 53(45S):93-97; Horneret al. (1998) Cell Immunol. 190:77-82; Jakob et al. (1998) J. Immunol.161:3042-3049; Redford et al. (1998) J. Immunol. 161:3930-3935; Weeratnaet al. (1998) Antisense & Nucleic Acid Drug Development 8:351-356;McCluskie et al. (1998) J. Immunol. 161(9):4463-4466; Gramzinski et al.(1998) Mol. Med. 4:109-118; Liu et al. (1998) Blood 92:3730-3736;Moldoveanu et al. (1998) Vaccine 16: 1216-1224; Brazolot Milan et al.(1998) Proc. Natl. Acad. Sci. USA 95:15553-15558; Briode et al. (1998)J. Immunol. 161:7054-7062; Briode et al. (1999) Int. Arch. AllergyImmunol. 118:453-456; Kovarik et al. (1999) J. Immunol. 162:1611-1617;Spiegelberg et al. (1999) Pediatr. Pulmonol. Suppl. 18:118-121;Martin-Orozco et al. (1999) Int. Immunol. 11:1111-1118; EP 468,520; WO96/02555; WO 97/28259; WO 98/16247; WO 98/18810; WO 98/37919; WO98/40100; WO 98/52581; WO 98/55495; WO 98/55609 and WO 99/11275. Seealso Elkins et al. (1999) J. Immunol. 162:2291-2298, WO 98/52962, WO99/33488, WO 99/33868, WO 99/51259 and WO 99/62923. See also Zimmermannet al. (1998)J. Immunol. 160:3627-3630; Krieg (1999) Trends Microbiol.7:64-65 and U.S. Pat. Nos. 5,663,153, 5,723,335 and 5,849,719. See alsoLiang et al. (1996) J. Clin. Invest. 98:1119-1129; Bohle et al. (1999)Eur. J. Immunol. 29:2344-2353 and WO 99/56755. See also WO 99/61056; WO00/06588; WO 00/16804; WO 00/21556; WO 00/54803; WO 00/61151; WO00/67023; WO 00/67787 and U.S. Pat. No. 6,090,791. In one embodiment, atleast one nucleic acid moiety of a CIC comprises a TG sequence or apyrimidine-rich (e.g., T-rich or C-rich) sequence, as described in PCTpublication WO 01/22972.

In some embodiments, the nucleic acid moiety is other than one or moreof the hexamers 5′-GACGTT-3′, 5′-GAGCTT-3′, 5′-TCCGGA-3′, 5′-AACGTT-3′,5′-GACGTT-3′, 5′-TACGTT-3′, 5′-CACGTT-3′, 5′-AGCGTT-3′, 5′-ATCGTT-3′,5′-ACCGTT-3′, 5′-AACGGT-3′, 5′-AACGAT-3′, 5′-AACGCT-3′, 5′-AACGTG-3′,5′-AACGTA-3′, and 5′-AACGTC-3′.

In some embodiments, the CIC contains at least 3, at least 10, at least30 or at least 100 nucleic acid moieties having a sequence describedabove.

C. Nucleic Acid Moiety Sequences: Heterogeneity and Position

It is contemplated that in a CIC comprising multiple nucleic acidmoieties, the nucleic acid moieties can be the same or different.

In one embodiment, all of the nucleic acid moieties in a CIC have thesame sequence. In one embodiment, a CIC comprises nucleic acid moietieswith at least 2, at least 3, at least 4, at least 5, or at least 6 ormore different sequences. In one embodiment, a CIC has fewer than 10different nucleic acid moieties. In one embodiment each of the nucleicacid moieties in a CIC has a different sequence.

In some embodiments, a single nucleic acid moiety contains more than oneiteration of a sequence motif listed above in §3(B), or two or moredifferent sequence motifs. The motifs within a single nucleic acidmoiety can be adjacent, overlapping, or separated by additionalnucleotide bases within the nucleic acid moiety. In an embodiment, anucleic acid moiety includes one or more palindromic regions. In thecontext of single-stranded oligonucleotides, the term “palindromic”refers to a sequence that would be palindromic if the oligonucleotidewere complexed with a complementary sequence to form a double-strandedmolecule. In another embodiment, one nucleic acid moiety has a sequencethat is palindromic or partially palindromic in relation to a secondnucleic acid moiety in the CIC. In an embodiment of the invention, thesequence of one or more of the nucleic acid moieties of a CIC is notpalindromic. In an embodiment of the invention, the sequence of one ormore of the nucleic acid moieties of a CIC does not include apalindromic sequence greater than four bases, optionally greater than 6bases.

As described supra, in various embodiments, one or more (e.g., all) ofthe nucleic acid moieties in a CIC comprises a 5′-CG-3′ sequence,alternatively a 5′-TCG-3′ sequence. In one embodiment, the nucleic acidmoiety is 5, 6 or 7 bases in length. In an embodiment, the nucleic acidmoiety has the formula 5′-TCG[(X)₂₋₄]-3′ or 5′-TCG(A/T)[(X)₁₋₃] or5′-TCG(A/T)CG(A/T)-3′ or 5′-TCGACGT-3′ (where each X is an independentlyselected nucleotide). In one embodiment, the aforementioned nucleic acidmoiety is a 5-prime moiety.

In one embodiment, a nucleic acid moiety comprises a sequence5′-TCGTCGA-3′. In one embodiment, a nucleic acid moiety comprises asequence selected from (all 5′43 3): TCGXXXX, TCGAXXX, XTCGXXX, XTCGAXX,TCGACGT, TCGAACG, TCGAGAT, TCGACTC, TCGAGCG, TCGATTT, TCGCTTT, TCGGTTT,TCGTTT, TCGTCGT, ATCGATT, TTCGTTT, TTCGATT, ACGTTCG, AACGTTC, TGACGTT,TGTCGTT, TCGXXX, TCGAXX, GTCGTT, GACGTT, ATCGAT, TCGTCG; TCGTTT; TCGAGA;TTCGAG; TTCGTT; AACGTT; AACGTTCG; AACGUTCG, ABGUTCG, TCGXX, TCGAX,TCGAT, TCGTT, TCGTC; TCGA, TCGT, and TCGX (where X is A, T, G or C; U is2′-deoxyuridine and B is 5-bromo-2′-deoxycytidine).

In one embodiment, the nucleic acid moiety is a 7-mer having thesequence TCGXXXX, TCGAXXX, XTCGXXX, XTCGAXX, TCGTCGA, TCGACGT, TCGAACG,TCGAGAT, TCGACTC, TCGAGCG, TCGATTT, TCGCTTT, TCGGTTT, TCGTTTT, TCGTCGT,ATCGATT, TTCGTTT; TTCGATT, ACGTTCG, AACGTTC, TGACGTT, or TGTCGTT; or isa 6-mer having the sequence TCGXXX, TCGAXX, TCGTCG, AACGTT, ATCGAT,GTCGTT, or GACGTT; or is a 5-mer having the sequence TCGXX, TCGAX,TCGAT, TCGTT, or TCGTC; or is a 4-mer having the sequence TCGA, TCGT, orTCGX, or is a 3-mer having the sequence TCG, where X is A, T, G or C.

In one embodiment, at least about 25%, preferably at least about 50%, orat least about 75%, and sometimes all of the nucleic acid moieties inthe CIC comprise at least one of the aforementioned sequences. In oneembodiment, at least one nucleic acid moiety does not comprise a CGmotif. In other embodiments, at least about 25%, sometimes at leastabout 50%, and sometimes at least about 75% of the nucleic acid moietiesin the CIC are nucleic acid moieties that do not have a CG motif or,alternatively, a TCG motif.

The position of a sequence or sequence motif in a CIC can influence theimmunomodulatory activity of the CIC, as is illustrated in the Examples,infra. In referring to the position of a sequence motif in a nucleicacid moiety of a CIC, the following terminology can be used: (1) In aCIC containing multiple nucleic acid moieties, a moiety with a free-5′end is referred to as “a 5-prime moiety.” It will be appreciated that asingle CIC may have multiple 5-prime moieties. (2) Within any particularnucleic acid moiety, a sequence or motif is in “the 5-prime position” ofthe moiety when there are no nucleotide bases 5′ to the referencesequence in that moiety. Thus, in the moiety with the sequence5′-TCGACGT-3′, the sequences T, TC, TCG and TCGA, are in “the 5-primeposition,” while the sequence GAC is not. By way of illustration, a CICcontaining the sequence TCG in the 5-prime position of a nucleic acidmoiety can render the CIC more active than an otherwise similar CIC witha differently positioned TCG motif. A CIC with a TCG sequence in a5-prime moiety, e.g., at the 5-prime position of the 5-prime moiety canrender a CIC particularly active. See e.g. Example 38. A nucleic acidmoiety with a free 5′ end can be designated using the symbol “5′^(F)” tothe left of the formula for the base sequence of the nucleic acid moiety(e.g., 5′^(F)-TACG-3′). As used herein, the term “free 5′ end” in thecontext of a nucleic acid moiety has its usual meaning and means thatthe 5′ terminus of the nucleic acid moiety is not conjugated to ablocking group or a non-nucleotide spacer moiety. The free 5′-nucleosidecontains an unmodified 5′-hydroxy group or a 5′-phosphate,5′-diphosphate, or 5′-triphosphate group, or other common modifiedphosphate groups (such as thiophosphate, dithiophosphate, and the like)that is not further linked to a blocking or functional group.

Immunostimulatory activity can also be influenced by the position of aCG motif in a nucleic acid moiety (e.g., in a 5′-moiety). For example,in one embodiment the CIC contains at least one nucleic acid moiety withthe sequence 5′-X-CG-Y-3′ where X is zero, one, or two nucleotides and Yis 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15nucleotides in length. In an embodiment, the 5′-X-CG-Y-3′ sequence is ina 5′-moiety of the CIC, e.g., the 5-prime position of the CIC. In anembodiment, the CIC contains 2, 3 or more nucleic acid moieties with asequence having the formula 5′-X-CG-Y-3′ sequence. For example, in anembodiment, all of the nucleic acid moieties of the CIC have sequencesof the formula 5′-X-CG-Y-3′ sequence.

Similarly, a CIC including the sequence TCGA (e.g., a sequence includingTCGACGT) in a nucleic acid moiety has immunomodulatory activity, and iseffective in IFN-α induction. A TCGA (e.g., a sequence includingTCGACGT) in a 5-prime moiety, e.g., at the 5-prime position of the5-prime moiety, renders the CIC particularly active. See examples 38 and49. Thus, in one embodiment, a CIC comprises a core structure with theformula (5′-N₁-3′)-S₁—N₂ (Ia) where N₁ has the sequence 5′-TCGAX-3′ andX is 0 to 20 nucleotide bases, often 0 to 3 bases. In one embodiment, Xis CGT. The sequence TCGTCGA is also particularly effective in IFN-αinduction.

In addition, the presence of free (unconjugated) nucleic acid 5′-endscan affect immunostimulatory activity. See, e.g., Example 39. In variousembodiments, a CIC of the invention comprises at least 1, at least 2, atleast 3, at least 4, or at least 5 free 5′ ends. In some embodiments,the number of free 5′-ends is from 1 to 10, from 2 to 6, from 3 to 5, orfrom 4-5. In one embodiment, the number of free 5′ ends is at leastabout 50 or at least about 100.

D. “Isolated Immunomodulatory Activity”

One property of a nucleic acid moiety is the “isolated immunomodulatoryactivity” associated with the nucleotide sequence of the nucleic acidmoiety. As noted supra, the present inventors have discovered that,surprisingly, CICs exhibit immunomodulatory activity even when none ofthe nucleic acid moieties of the CIC has a sequence that, if presentedas a polynucleotide alone, exhibits comparable immunomodulatoryactivity.

In some embodiments, a nucleic acid moiety of a CIC does not have“isolated immunomodulatory activity,” or has “inferior isolatedimmunomodulatory activity,” (i.e., when compared to the CIC), asdescribed below.

The “isolated immunomodulatory activity” of a nucleic acid moiety isdetermined by measuring the immunomodulatory activity of an isolatedpolynucleotide having the primary sequence of the nucleic acid moiety,and having the same nucleic acid backbone (e.g., phosphorothioate,phosphodiester, chimeric). For example, a CIC having the structure“5′-TCGTCG-3′-HEG-5′-ACG′TTCG-3′-HEG-5′-AGATGAT-3” contains threenucleic acid moieties. To determine the independent immunomodulatoryactivity of, for example, the first nucleic acid moiety in the CIC, atest polynucleotide having the same sequence (i.e., 5′-TCGTCG-3′) andsame backbone structure (e.g., phosphorothioate) is synthesized usingroutine methods, and its immunomodulatory activity (if any) is measured.Immunomodulatory activity can be determined using standard assays whichindicate various aspects of the immune response, such as those describedin §2, supra. Preferably the human PBMC assay described in §2, supra, isused. As discussed supra, to account for donor variation, typically theassay is carried out using cells obtained from multiple donors. Apolynucleotide does not have immunomodulatory activity (and thecorresponding nucleic acid moiety does not have “isolatedimmunomodulatory activity”) when the amount of IFN-γ secreted by PBMCscontacted with the polynucleotide is not significantly greater (e.g.,less than about 2-fold greater) in the majority of donors than in theabsence of the test compound or, (in some embodiments) in the presenceof an inactive control compound (e.g., 5′-TGACTGTGAACCTTAGAGATGA-3′ (SEQID NO:3).)

To compare the immunomodulatory activity of a CIC and an isolatedpolynucleotide, immunomodulatory activity is measured, preferably usingthe human PBMC assay described in §2, supra. Usually, the activity oftwo compounds is compared by assaying them in parallel under the sameconditions (e.g., using the same cells), usually at a concentration ofabout 20 μg/ml. As noted supra, typically, concentration is determinedby measuring absorbance at 260 nm and using the conversion 0.5OD₂₆₀/ml=20 μg/ml. This normalizes the amount of total nucleic acid inthe test sample. Alternatively, concentration or weight can be measuredby other methods known in the art. If desired, the amount of nucleicacid moiety can be determined by measuring absorbance at 260, and theweight of the CIC calculated using the molecular formula of the CIC.This method is sometimes used when the ratio of weight contributed bythe spacer moiety(s) to weight contributed by the nucleic acid moietiesin a CIC is high (i.e., greater than 1).

Alternatively, a concentration of 3 μM may be used, particularly whenthe calculated molecular weights of two samples being compared differ bymore than 20%.

A nucleic acid moiety of a CIC is characterized as having “inferiorimmunomodulatory activity,” when the test polynucleotide has lessactivity than the CIC to which it is compared. Preferably the isolatedimmunomodulatory activity of the test polynucleotide is no more thanabout 50% of the activity of the CIC, more preferably no more than about20%, most preferably no more than about 10% of the activity of the CIC,or in some embodiments, even less.

For CICs with multiple (e.g., multiple different) nucleic acid moieties,it is also possible to determine the immunomodulatory activity (if any)of a mixture of test polynucleotides corresponding to the multiplenucleic acid moieties. The assay can be carried out using a total amountof test polynucleotide (i.e., in the mixture) which equals the amount ofCIC used. Alternatively, an amount of each test polynucleotide, or eachdifferent test polynucleotide, in the mixture can be equal to the amountof the CIC in the assay. As noted in §2, to account for donor variation,preferably assays and analysis use PMBCs from multiple donors.

In one embodiment, one or more (e.g., at least about 2, at least about4, or at least about 25%, at least about 50%, or all, measuredindividually or, alternatively, in combination) of the nucleic acidmoieties in a CIC do not have isolated immunomodulatory activity. In oneembodiment, one or more (e.g., at least about 2, at least about 4, or atleast about 25%, at least about 50%, or all, measured individually or,alternatively, in combination) has inferior isolated immunomodulatoryactivity compared to the CIC.

In a related embodiment, a CIC comprises one or more nucleic acidmoieties with isolated immunomodulatory activity. For example, in someembodiments, all or almost all (e.g., at least 90%, preferably at least95%) of the nucleic acid moieties has isolated immunomodulatoryactivity. For example, a CIC comprising a multivalent spacer(s) cancomprise more than 4, often more than 10, frequently at least about 20,at least about 50, at least about 100, at least about 400 or at leastabout 1000 nucleic acid moieties (e.g., at least about 2500) withisolated immunostimulatory activity (e.g., having the sequence 5′-TGACTG TGA ACG TTC GAG ATG A-3′ (SEQ ID NO:2)).

Thus, in a particular CIC, the number of nucleic acid moieties that haveisolated immunomodulatory activity can be zero (0), one (1), 2 or more,3 or more, fewer than 3, 4 or more, fewer than 4, 5 or more, fewer than5, at least 10, at least about 20, at least about 50, at least about100, at least about 400 or at least about 1000, all, or less than all,of the nucleic acid moieties of the CIC.

E. Structure of the Nucleic Acid Moiety

A nucleic acid moiety of a CIC may contain structural modificationsrelative to naturally occurring nucleic acids. Modifications include anyknown in the art for polynucleotides, but are not limited to,modifications of the 3′OH or 5′OH group, modifications of the nucleotidebase, modifications of the sugar component, and modifications of thephosphate group. Various such modifications are described below.

The nucleic acid moiety may be DNA, RNA or mixed DNA/RNA, singlestranded, double stranded or partially double stranded, and may containother modified polynucleotides. Double stranded nucleic acid moietiesand CICs are contemplated, and the recitation of the term “base” or“nucleotide” is intended to encompass basepair or basepaired nucleotide,unless otherwise indicated. A nucleic acid moiety may containnaturally-occurring or modified, non-naturally occurring bases, and maycontain modified sugar, phosphate, and/or termini. For example,phosphate modifications include, but are not limited to, methylphosphonate, phosphorothioate, phosphoramidate (bridging ornon-bridging), phosphotriester and phosphorodithioate and may be used inany combination. Other non-phosphate linkages may also be used.Preferably, CICs and nucleic acid moieties of the present inventioncomprise phosphorothioate backbones. Sugar modifications known in thefield, such as 2′-alkoxy-RNA analogs, 2′-amino-RNA analogs and2′-alkoxy- or amino-RNA/DNA chimeras and others described herein, mayalso be made and combined with any phosphate modification. Examples ofbase modifications (discussed further below) include, but are notlimited to, addition of an electron-withdrawing moiety to C-5 and/or C-6of a cytosine (e.g., 5-bromocytosine, 5-chlorocytosine,5-fluorocytosine, 5-iodocytosine) and C-5 and/or C-6 of a uracil (e.g.,5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil). See, forexample, PCT Application No. WO 99/62923.

The nucleic acid moiety can also contain phosphate-modified nucleotides.Synthesis of nucleic acids containing modified phosphate linkages ornon-phosphate linkages is also know in the art. For a review, seeMatteucci (1997) “Oligonucleotide Analogs: an Overview” inOligonucleotides as Therapeutic Agents, (D. J. Chadwick and G. Cardew,ed.) John Wiley and Sons, New York, N.Y. The phosphorous derivative (ormodified phosphate group) which can be attached to the sugar or sugaranalog moiety in the nucleic acids of the present invention can be amonophosphate, diphosphate, triphosphate, alkylphosphonate,phosphorothioate, phosphorodithioate, phosphoramidate or the like. Thepreparation of the above-noted phosphate analogs, and theirincorporation into nucleotides, modified nucleotides andoligonucleotides, per se, is also known and need not be described herein detail. Peyrottes et al. (1996) Nucleic Acids Res. 24:1841-1848;Chaturvedi et al. (1996) Nucleic Acids Res. 24:2318-2323; and Schultz etal. (1996) Nucleic Acids Res. 24:2966-2973. For example, synthesis ofphosphorothioate oligonucleotides is similar to that described above fornaturally occurring oligonucleotides except that the oxidation step isreplaced by a sulfurization step (Zon (1993) “OligonucleosidePhosphorothioates” in Protocols for Oligonucleotides and Analogs,Synthesis and Properties (Agrawal, ed.) Humana Press, pp. 165-190).Similarly the synthesis of other phosphate analogs, such asphosphotriester (Miller et al. (1971) JACS 93:6657-6665), non-bridgingphosphoramidates (Jager et al. (1988) Biochem. 27:7247-7246), N3′ to P5′phosphoramidates (Nelson et al. (1997) JOC 62:7278-7287) andphosphorodithioates (U.S. Pat. No. 5,453,496) has also been described.Other non-phosphorous based modified nucleic acids can also be used(Stirchak et al. (1989) Nucleic Acids Res. 17:6129-6141). Nucleic acidswith phosphorothioate backbones appear to be more resistant todegradation after injection into the host. Braun et al. (1988) J.Immunol. 141:2084-2089; and Latimer et al. (1995) Mol. Immunol.32:1057-1064.

Nucleic acid moieties used in the invention can comprise ribonucleotides(containing ribose as the only or principal sugar component), and/ordeoxyribonucleotides (containing deoxyribose as the principal sugarcomponent). Modified sugars or sugar analogs can be incorporated in thenucleic acid moiety. Thus, in addition to ribose and deoxyribose, thesugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose,arabinose, xylose, lyxose, and a sugar “analog” cyclopentyl group. Thesugar can be in pyranosyl or in a furanosyl form. The sugar moiety ispreferably the furanoside of ribose, deoxyribose, arabinose or2′-0-alkylribose, and the sugar can be attached to the respectiveheterocyclic bases either in α or β anomeric configuration. Sugarmodifications include, but are not limited to, 2′-alkoxy-RNA analogs,2′-amino-RNA analogs and 2′-alkoxy- or amino-RNA/DNA chimeras. Forexample, a sugar modification in the CIC includes, but is not limitedto, 2′-amino-2′-deoxyadenosine. The preparation of these sugars or sugaranalogs and the respective “nucleosides” wherein such sugars or analogsare attached to a heterocyclic base (nucleic acid base) per se is known,and need not be described here, except to the extent such preparationcan pertain to any specific example. Sugar modifications may also bemade and combined with any phosphate modification in the preparation ofa CIC.

The heterocyclic bases, or nucleic acid bases, which are incorporated inthe nucleic acid moiety can be the naturally-occurring principal purineand pyrimidine bases, (namely uracil, thymine, cytosine, adenine andguanine, as mentioned above), as well as naturally-occurring andsynthetic modifications of said principal bases.

Those skilled in the art will recognize that a large number of“synthetic” non-natural nucleosides comprising various heterocyclicbases and various sugar moieties (and sugar analogs) are available inthe art, and that as long as other criteria of the present invention aresatisfied, the nucleic acid moiety can include one or severalheterocyclic bases other than the principal five base components ofnaturally-occurring nucleic acids. Preferably, however, the heterocyclicbase is, without limitation, uracil-5-yl, cytosin-5-yl, adenin-7-yl,adenin-8-yl, guanin-7-yl, guanin-8-yl,4-aminopyrrolo[2,3-d]pyrimidin-5-yl,2-amino-4-oxopyrolo[2,3-d]pyrimidin-5-yl, or2-amino-4-oxopyrrolo[2,3-d]pyrimidin-3-yl groups, where the purines areattached to the sugar moiety of the nucleic acid moiety via the9-position, the pyrimidines via the 1-position, the pyrrolopyrimidinesvia the 7-position and the pyrazolopyrimidines via the 1-position.

The nucleic acid moiety may comprise at least one modified base. As usedherein, the term “modified base” is synonymous with “base analog”, forexample, “modified cytosine” is synonymous with “cytosine analog.”Similarly, “modified” nucleosides or nucleotides are herein defined asbeing synonymous with nucleoside or nucleotide “analogs.” Examples ofbase modifications include, but are not limited to, addition of anelectron-withdrawing moiety to C-5 and/or C-6 of a cytosine of thenucleic acid moiety. Preferably, the electron-withdrawing moiety is ahalogen. Such modified cytosines can include, but are not limited to,azacytosine, 5-bromocytosine, 5-chlorocytosine, chlorinated cytosine,cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine,5,6-dihydrocytosine, 5-iodocytosine, 5-nitrocytosine, and any otherpyrimidine analog or modified pyrimidine. Other examples of basemodifications include, but are not limited to, addition of anelectron-withdrawing moiety to C-5 and/or C-6 of a uracil of the nucleicacid moiety. Preferably, the electron-withdrawing moiety is a halogen.Such modified uracils can include, but are not limited to,5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil. Also see,Kandimalla et al., 2001, Bioorg. Med. Chem. 9:807-13.

Other examples of base modifications include the addition of one or morethiol groups to the base including, but not limited to, 6-thio-guanine,4-thio-thymine and 4-thio-uracil.

The preparation of base-modified nucleosides, and the synthesis ofmodified oligonucleotides using said base-modified nucleosides asprecursors, has been described, for example, in U.S. Pat. Nos.4,910,300, 4,948,882, and 5,093,232. These base-modified nucleosideshave been designed so that they can, be incorporated by chemicalsynthesis into either terminal or internal positions of anoligonucleotide. Such base-modified nucleosides, present at eitherterminal or internal positions of an oligonucleotide, can serve as sitesfor attachment of a peptide or other antigen. Nucleosides modified intheir sugar moiety have also been described (including, but not limitedto, e.g., U.S. Pat. Nos. 4,849,513, 5,015,733, 5,118,800, 5,118,802) andcan be used similarly.

4. Non-Nucleic Acid Spacer Moieties

The CIC compounds of the invention comprise one or more non-nucleic acidspacer moieties covalently bound to the nucleic acid moieties. Forconvenience, non-nucleic acid spacer moieties are sometimes referred toherein simply as “spacers” or “spacer moieties.”

Spacers are generally of molecular weight about 50 to about 500,000(e.g. about 50 to about 50,000), sometimes from about 75 to about 5000,sometimes from about 75 to about 500, which are covalently bound, invarious embodiments, to one, two, three, or more than three nucleic acidmoieties. A variety of agents are suitable for connecting nucleic acidmoieties. For example, a variety of compounds referred to in thescientific literature as “non-nucleic acid linkers,” “non-nucleotidiclinkers,” or “valency platform molecules” may be used as spacers in aCIC. A spacer moiety is said to comprise a particular spacer component(e.g., hexaethylene glycol) when the spacer includes the component (or asubstituted derivative) as a subunit or portion of the spacer. Forexample, the spacer shown in Example 49 can be described as comprising apolysaccharide component, a hexaethylene glycol component, and aderivatized thioether linker component. As described infra, in certainembodiments, a spacer comprises multiple covalently connected subunitsand may have a homopolymeric or heteropolymeric structure. Often thesubunits are connected by a linker, phosphodiester linkage, and/orphosphorothioate ester linkage. See the Examples, infra. Nonnucleotidespacer moieties of a CIC comprising or derived from such multiple unitscan be referred to as “compound spacers.” In one embodiment, forillustration and not limitation, the CIC comprises a compound spacercomprising any two or more (e.g., 3 or more, 4 or more, or 5 or more) ofthe following compounds in phosphodiester linkage and/orphosphorothioate ester linkage: oligoethylene glycol unit (e.g.,triethylene glycol spacer; hexaethylene glycol spacer); alkyl unit(e.g., propyl spacer; butyl spacer; hexyl spacer);2-(hydroxymethyl)ethyl spacer; glycerol spacer; trebler spacer;symmetrical doubler spacer.

It will be appreciated that mononucleosides and polynucleotides are notincluded in the definition of non-nucleic acid spacers, without whichexclusion there would be no difference between nucleic acid moiety andan adjacent non-nucleic acid spacer moiety.

A variety of spacers are described herein, for illustration and notlimitation. It will be appreciated by the reader that, for convenience,a spacer moiety (or component of a spacer moiety) is sometimes referredto by the chemical name of the compound (e.g., hexaethylene glycol) fromwhich the spacer moiety or component is derived, with the understandingthat the CIC actually comprises the conjugate of the compound(s) tonucleic acid moieties. As will be understood by the ordinarily skilledpractitioner (and as described in greater detail hereinbelow), thenon-nucleic acid spacer can be (and usually is) formed from a spacermoiety precursor(s) that include reactive groups to permit coupling ofone more nucleic acid (e.g., oligonucleotides) to the spacer moietyprecursor to form the CIC and protecting groups may be included. Thereactive groups on the spacer precursor may be the same or different.

Exemplary non-nucleic acid spacers comprise oligo-ethylene glycol (e.g.,triethylene glycol, tetraethylene glycol, hexaethylene glycol spacers,and other polymers comprising up to about 10, about 20, about 40, about50, about 100 or about 200 ethylene glycol units), alkyl spacers (e.g.,propyl, butyl, hexyl, and other C2-C12 alkyl spacers, e.g., usuallyC2-C10 alkyl, most often C2-C6 alkyl), symmetric or asymmetric spacersderived from glycerol, pentaerythritol, 1,3,5-trihydroxycyclohexane or1,3-diamino-2-propanol (e.g., symmetrical doubler and trebler spacermoieties described herein). Optionally these spacer components aresubstituted. For example, as will be understood by one of ordinary skillin the art, glycerol and 1,3-diamino-2-propanol may be substituted atthe 1, 2, and/or 3 position (e.g., replacement of one or more hydrogensattached to carbon with one of the groups listed below). Similarly,pentaerythritol may be substituted at any, or all, of the methylenepositions with any of the groups described below. Substituents includealcohol, alkoxy (such as methoxy, ethoxy, and propoxy), straight orbranched chain alkyl (such as C1-C12 alkyl, preferably C1-C10 alkyl),amine, aminoalkyl (such as amino C1-C12 alkyl, preferably amino C1-C10alkyl), phosphoramidite, phosphate, phosphoramidate, phosphorodithioate,thiophosphate, hydrazide, hydrazine, halogen, (such as F, Cl, Br, or I),amide, alkylamide (such as amide C1-C12 alkyl, preferably C1-C10 alkyl),carboxylic acid, carboxylic ester, carboxylic anhydride, carboxylic acidhalide, ether, sulfonyl halide, imidate ester, isocyanate,isothiocyanate, haloformate, carbodiimide adduct, aldehydes, ketone,sulfhydryl, haloacetyl, alkyl halide, alkyl sulfonate, NR1R2 whereinR1R2 is —C(═O)CH═CHC(═O) (maleimide), thioether, cyano, sugar (such asmannose, galactose, and glucose), α,β-unsaturated carbonyl, alkylmercurial, α,β-unsaturated sulfone.

In one embodiment, a spacer may comprise one or more abasic nucleotides(i.e., lacking a nucleotide base, but having the sugar and phosphateportions). Exemplary abasic nucleotides include 1′2′-dideoxyribose,1′-deoxyribose, 1′-deoxyarabinose and polymers thereof.

Spacers can comprise heteromeric or homomeric oligomers and polymers ofthe nonnucleic acid components described herein (e.g., linked by aphosphodiester or phosphorothioate linkage or, alternatively an amide,ester, ether, thioether, disulfide, phosphoramidate, phosphotriester,phosphorodithioate, methyl phosphonate or other linkage). For example,in one embodiment, the spacer moiety comprises a branched spacercomponent (e.g., glycerol) conjugated via a phosphodiester orphosphorothioate linkage to an oligoethylene glycol such as HEG (see,e.g., C-94). Another example, is a spacer comprising a multivalentspacer component conjugated to an oligoethylene glycol such as HEG.

Other suitable spacers comprise substituted alkyl, substitutedpolyglycol, optionally substituted polyamine, optionally substitutedpolyalcohol, optionally substituted polyamide, optionally substitutedpolyether, optionally substituted polyimine, optionally substitutedpolyphosphodiester (such as poly(1-phospho-3-propanol), and the like.Optional substituents include alcohol, alkoxy (such as methoxy, ethoxy,and propoxy), straight or branched chain alkyl (such as C1-C12 alkyl,preferably C1-C12 alkyl), amine, aminoalkyl (such as amino C1-C12 alkyl,preferably C1-C12 alkyl), phosphoramidite, phosphate, thiophosphate,hydrazide, hydrazine, halogen, (such as F, Cl, Br, or I), amide,alkylamide (such as amide C1-C12 alkyl or C1-C12 alkyl), carboxylicacid, carboxylic ester, carboxylic anhydride, carboxylic acid halide,ether, sulfonyl halide, imidate ester, isocyanate, isothiocyanate,haloformate, carbodiimide adduct, aldehydes, ketone, sulfhydryl,haloacetyl, alkyl halide, alkyl sulfonate, NR1R2 wherein R1R2 is—C(═O)CH═CHC(═O) (maleimide), thioether, cyano, sugar (such as mannose,galactose, and glucose), α,β-unsaturated carbonyl, alkyl mercurial,α,β-unsaturated sulfone.

Other suitable spacers may comprise polycyclic molecules, such as thosecontaining phenyl or cyclohexyl rings. The spacer may be a polyethersuch as polyphosphopropanediol, polyethylene glycol, polypropyleneglycol, a bifunctional polycyclic molecule such as a bifunctionalpentalene, indene, naphthalene, azulene, heptalene, biphenylene,asymindacene, sym-indacene, acenaphthylene, fluorene, phenalene,phenanthrene, anthracene, fluoranthene, acephenanthrylene,aceanthrylene, triphenylene, pyrene, chrysene, naphthacene, thianthrene,isobenzofuran, chromene, xanthene, phenoxathiin, which may besubstituted or modified, or a combination of the polyethers and thepolycyclic molecules. The polycyclic molecule may be substituted orpolysubstituted with C1-C5 alkyl, C6 alkyl, alkenyl, hydroxyalkyl,halogen or haloalkyl group. Nitrogen-containing polyheterocyclicmolecules (e.g., indolizine) are typically not suitable spacers. Thespacer may also be a polyalcohol, such as glycerol or pentaerythritol.In one embodiment, the spacer comprises (1-phosphopropane)₃-phosphate or(1-phosphopropane)₄-phosphate (also called tetraphosphopropanediol andpentaphosphopropanediol). In one embodiment, the spacer comprisesderivatized 2,2′-ethylenedioxydiethylamine (EDDA).

Other examples of non-nucleic acid spacers that may be used in CICsinclude “linkers” described by Cload and Schepartz, J. Am. Chem. Soc.(1991), 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. (1991),113:5109; Ma et al., Nucleic Acids Research (1993), 21:2585; Ma et al.,Biochemistry (1993), 32:1751; McCurdy et al., Nucleosides &Nucleotides(1991), 10:287; Jaschke et al., Tetrahedron Lett. (1993), 34:301; Ono etal., Biochemistry (1991), 30:9914; and Arnold et al., InternationalPublication No. WO 89/02439 and EP0313219B1 entitled “Non-nucleic acidLinking Reagents for Nucleotide Probes,” linkers described by Salunkheet al., J. Am. Chem. Soc. (1992), 114:8768; Nelson et al., Biochemistry35:5339-5344 (1996); Bartley et al., Biochemistry 36:14502-511 (1997);Dagneaux et al. Nucleic Acids Research 24:4506-12 (1996); Durand et al.,Nucleic Acids Research 18:6353-59 (1990); Reynolds et al., Nucleic AcidsResearch, 24:760-65 (1996); Hendry et al. Biochemica et Biophysica Acta,1219:405-12 (1994); Altmann et al., Nucleic Acids Research, 23:4827-35(1995), and U.S. Pat. No. 6,117,657 (Usman et al.).

Suitable spacer moieties can contribute charge and/or hydrophobicity tothe CIC, contribute favorable pharmacokinetic properties (e.g., improvedstability, longer residence time in blood) to the CIC, and/or result intargeting of the CIC to particular cells or organs. Spacer moieties canbe selected or modified to tailor the CIC for desired pharmacokineticproperties, induction of a particular immune response, or suitabilityfor desired modes of administration (e.g., oral administration).

In a CIC comprising more than one spacer moiety, the spacers may be thesame or different. Thus, in one embodiment all of the non-nucleic acidspacer moieties in a CIC have the same structure. In one embodiment, aCIC comprises non-nucleic acid spacer moieties with at least 2, at least3, at least 4, at least 5, or at least 6 or more different structures.

In some contemplated embodiments of the invention, the spacer moiety ofa CIC is defined to exclude certain structures. Thus, in someembodiments of the invention, a spacer is other than an abasicnucleotide or polymer of abasic nucleotides. In some embodiments of theinvention, a spacer is other than a oligo(ethyleneglycol) (e.g., HEG,TEG and the like) or poly(ethyleneglycol). In some embodiments a spaceris other than a C3 alkyl spacer. In some embodiments a spacer is otherthan an alkyl or substituted spacer. In some embodiments, a spacer isother than a polypeptide. Thus, in some embodiments, an immunogenicmolecule, e.g., a protein or polypeptide, is not suitable as a componentof spacer moieties. However, as discussed infra, it is contemplated thatin certain embodiments, a CIC is a “proteinaceous CIC” i.e., comprisinga spacer moiety comprising a polypeptide (i.e., oligomer or polymer ofamino acids). For example, as discussed infra, in some embodiments, apolypeptide antigen can be used as a platform (multivalent spacer) towhich a plurality of nucleic acid moieties are conjugated. However, insome embodiments, the spacer moiety is not proteinaceous and/or is notan antigen (i.e., the spacer moiety, if isolated from the CIC, is not anantigen).

Suitable spacer moieties do not render the CIC of which they are acomponent insoluble in an aqueous solution (e.g., PBS, pH 7.0). Thus,the definition of spacers excludes microcarriers or nanocarriers. Inaddition, a spacer moiety that has low solubility, such as a dodecylspacer (solubility <5 mg/ml when measured as dialcohol precursor1,12-dihydroxydodecane) is not preferred because it can reduce thehydrophilicity and activity of the CIC. Preferably, spacer moieties havesolubility much greater than 5 mg/ml (e.g., solubility at least about 20mg/ml, at least about 50 mg/ml or at least about 100 mg/ml), e.g., whenmeasured as dialcohol precursors. The form of the spacer moiety used fortesting its water solubility is generally its most closely relatedunactivated and unprotected spacer precursor molecule. For example, C-19contains a spacer moiety including a dodecyl group with phosphorothioatediester linkages at the C-1 and C-12 positions, thereby connecting thespacer moiety to the nucleic acid moieties. In this case, the watersolubility of the dialcohol version of the dodecyl spacer,1,12-dihydroxydodecane, was tested and found to be less than 5 mg/ml.Spacers with higher water solubility, when tested as their dialcoholprecursors, resulted in more immunostimulatory CICs. These higher watersolubility spacers include, without limitation, propane 1,3 diol;glycerol; butane-1,4-diol; pentane-1,5-diol; hexane-1,6-diol;triethylene glycol, tetraethylene glycol and HEG.

A. Charged and Multiunit Spacer Moieties

The charge of a CIC may be contributed by phosphate, thiophosphate, orother groups in the nucleic acid moieties as well as groups innon-nucleic acid spacer moieties. In some embodiments of the invention,a non-nucleic acid spacer moiety carries a net charge (e.g., a netpositive charge or net negative charge when measured at pH 7). In oneembodiment, the CIC has a net negative charge. In some embodiments, thenegative charge of a spacer moiety in a CIC is increased by derivatizinga spacer subunit described herein to increase its charge. For example,glycerol can be covalently bound to two nucleic acid moieties and theremaining alcohol can be reacted with an activated phosphoramidite,followed by oxidation or sulfurization to form a phosphate orthiophosphate, respectively. In certain embodiments the negative chargecontributed by the non-nucleic acid spacer moieties in a CIC (i.e., thesum of the charges when there is more than one spacer) is greater thanthe negative charge contributed by the nucleic acid moieties of the CIC.Charge can be calculated based on molecular formula, or determinedexperimentally, e.g., by capillary electrophoresis (Li, ed., 1992,Capillary Electrophoresis, Principles, Practice and Application ElsevierScience Publishers, Amsterdam, The Netherlands, pp 202-206).

As is noted supra, suitable spacers include polymers of smallernon-nucleic acid (e.g., non-nucleotide) compounds that may be used asspacers. The smaller non-nucleic acid compounds include compoundscommonly referred to as non-nucleotide “linkers” and other spacersdescribed herein. Such polymers (i.e., “multiunit spacers”) may beheteromeric or homomeric, and often comprise monomeric units (e.g.,oligoethylene glycols, [e.g., HO—(CH2CH2-O)_(N)—H, where N=2-10; such asHEG and TEG], glycerol, 1′2′-dideoxyribose, and the like) linked by anester linkage (e.g., phosphodiester or phosphorothioate ester). Thus, inone embodiment the spacer comprises a polymeric (e.g., heteropolymeric)structure of non-nucleotide units (e.g., from 2 to about 100 units,alternatively 2 to about 50, e.g., 2 to about 5, alternatively e.g.,about 5 to about 50, e.g., about 5 to about 20).

For illustration, CICs containing multiunit spacers include

5′-TCGTCG-(C3)₁₅-T 5′-TCGTCG-(glycerol)₁₅-T 5′-TCGTCG-(TEG)₈-T5′-TCGTCG-(HEG)₄-Twhere (C3)₁₅ means 15 propyl linkers connected via phosphorothioateesters; (glycerol)₁₅ means 15 glycerol linkers connected viaphosphorothioate esters; (TEG)₈ means 8 triethyleneglycol linkersconnected via phosphorothioate esters; and (HEG)₄ means 4hexaethyleneglycol linkers connected via phosphorothioate esters. Itwill be appreciated that certain multiunit spacers have a net negativecharge, and that the negative charge can be increased by increasing thenumber of e.g., ester-linked monomeric units.

B. Multivalent Spacer Moiety

In certain embodiments, a spacer moiety is a multivalent non-nucleicacid spacer moiety (i.e., a “multivalent spacer”). As used in thiscontext, a CIC containing a multivalent spacer contains a spacercovalently bound to three (3) or more nucleic acid moieties. Multivalentspacers are sometimes referred to in the art as “platform molecules.”Multivalent spacers can be polymeric or nonpolymeric. Examples ofsuitable molecules include glycerol or substituted glycerol (e.g.,2-hydroxymethyl glycerol, levulinyl-glycerol); tetraminobenzene,heptaminobetacyclodextrin, 1,3,5-trihydroxycyclohexane, pentaerythritoland derivatives of pentaerythritol, tetraminopentaerythritol,1,4,8,11-tetraazacyclo tetradecane (Cyclam),1,4,7,10-tetraazacyclododecane (Cyclen), polyethyleneimine,1,3-diamino-2-propanol and substituted derivatives (e.g., “symmetricaldoubler”), [propyloxymethyl]ethyl compounds (e.g., “trebler”),polyethylene glycol derivatives such as so-called “Star PEGs” and “bPEG”(see, e.g., Gnanou et al., 1988, Makromol. Chem. 189:2885; Rein et al.,1993, Acta Polymer 44:225, Merrill et al., U.S. Pat. No. 5,171,264;Shearwater Polymers Inc., Huntsville Ala.), dendrimers andpolysaccharides.

Dendrimers are known in the art and are chemically defined globularmolecules, generally prepared by stepwise or reiterative reaction ofmultifunctional monomers to obtain a branched structure (see, e.g.,Tomalia et al., 1990, Angew. Chem. Int. Ed. Engl. 29:138-75). A varietyof dendrimers are known, e.g., amine-terminated polyamidoamine,polyethyleneimine and polypropyleneimine dendrimers. Exemplarydendrimers for use in the present invention include “dense star”polymers or “starburst” polymers such as those described in U.S. Pat.Nos. 4,587,329; 5,338,532; and 6,177,414, including so-called“poly(amidoamine) (“PAMAM”) dendrimers.” Still other multimeric spacermolecules suitable for use within the present invention includechemically-defined, non-polymeric valency platform molecules such asthose disclosed in U.S. Pat. No. 5,552,391; and PCT applicationsPCT/US00/15968 (published as WO 00/75105); PCT/US96/09976 (published asWO 96/40197), PCT/US97/10075 (published as WO 97/46251); PCT/US94/10031(published as WO 95/07073); and PCT/US99/29339 (published as WO00/34231). Many other suitable multivalent spacers can be used and willbe known to those of skill in the art.

Conjugation of a nucleic acid moiety to a platform molecule can beeffected in any number of ways, typically involving one or morecrosslinking agents and functional groups on the nucleic acid moiety andplatform molecule. Linking groups are added to platforms using standardsynthetic chemistry techniques. Linking groups can be added to nucleicacid moieties using standard synthetic techniques.

Multivalent spacers with a variety of valencies may be used in thepractice of the invention, and in various embodiments the multivalentspacer of a CIC is bound to between about 3 and about 400 nucleic acidmoieties, sometimes about 100 to about 500, sometimes about 150 to about250, sometimes 3-200, sometimes from 3 to 100, sometimes from 3-50,frequently from 3-10, and sometimes more than 400 nucleic acid moieties.In various embodiments, the multivalent spacer is conjugated to morethan 10, more than 25, more than 50, more than 100 or more than 500nucleic acid moieties (which may be the same or different). It will beappreciated that, in certain embodiments in which a CIC comprises amultivalent spacer, the invention provides a population of CICs withslightly different molecular structures. For example, when a CIC isprepared using a dendrimer, polysaccharide or other multivalent spacerwith a high valency, a somewhat heterogeneous mixture of molecules isproduced, i.e., comprising different numbers (within or predominantlywithin a determinable range) of nucleic acid moieties joined to themultivalent spacer moiety. When a dendrimer, polysaccharide or the likeis used as an element of a multivalent spacer, the nucleic acid moietiescan be joined directly or indirectly to the element (e.g., dendrimer).For example, a CIC can comprise nucleic acid moiety joined to adendrimer via an oligoethyleneglycol element (where thedendrimer+oligoethyleneglycol constitute the spacer moiety). It will berecognized that the nucleic acid moieties may be conjugated to more thanone spacer moiety, as described in §III (1)B, supra.

Polysaccharides derivatized to allow linking to nucleic acid moietiescan be used as multivalent spacers in CICs. Suitable polysaccharides maybe naturally occurring polysaccharides or synthetic polysaccharides.Exemplary polysaccharides include, e.g., dextran, mannin, chitosan,agarose, and starch. Mannin may be used, for example, because there aremannin (mannose) receptors on immunologically relevant cell types, suchas monocytes and alveolar macrophages, and so the polysaccharide spacermoiety may be used for targeting particular cell types. In anembodiment, the polysaccharide is cross-linked. One suitable compound isepichlorohydrin-crosslinked sucrose (e.g., FICOLL®). FICOLL® issynthesized by cross-linking sucrose with epichlorohydrin which resultsin a highly branched structure. For example, as shown in Example 49,aminoethylcarboxymethyl-ficoll (AECM-Ficoll) can be prepared by themethod of Inman, 1975, J. Imm. 114:704-709. The number of nucleic acidmoieties in a CIC comprising a polysaccharide can be any range describedherein for a CIC (e.g., a multivalent CIC). For example, in oneembodiment, the polysaccharide comprises between about 150 and about 250nucleic acid moieties. AECM-Ficoll can then be reacted with aheterobifunctional crosslinking reagent, such as 6-maleimido caproicacyl N-hydroxysuccinimide ester, and then conjugated to athiol-derivatized nucleic acid moiety (see Lee, et al., 1980, Mol. Imm.17:749-56). Other polysaccharides may be modified similarly.

5. Synthesis of CICs

It will be well within the ability of one of skill, guided by thisspecification and knowledge in the art, to prepare CICs using routinemethods. Techniques for making nucleic acid moieties (e.g.,oligonucleotides and modified oligonucleotides) are known. Nucleic acidmoieties can be synthesized using techniques including, but not limitedto, enzymatic methods and chemical methods and combinations of enzymaticand chemical approaches. For example, DNA or RNA containingphosphodiester linkages can be chemically synthesized by sequentiallycoupling the appropriate nucleoside phosphoramidite to the 5′-hydroxygroup of the growing oligonucleotide attached to a solid support at the3′-end, followed by oxidation of the intermediate phosphite triester toa phosphate triester. Useful solid supports for DNA synthesis includeControlled Pore Glass (Applied Biosystems, Foster City, Calif.),polystyrene bead matrix (Primer Support, Amersham Pharmacia, Piscataway,N.J.) and TentGel (Rapp Polymere GmbH, Tubingen, Germany). Once thedesired oligonucleotide sequence has been synthesized, theoligonucleotide is removed from the support, the phosphate triestergroups are deprotected to phosphate diesters and the nucleoside basesare deprotected using aqueous ammonia or other bases.

For instance, DNA or RNA polynucleotides (nucleic acid moieties)containing phosphodiester linkages are generally synthesized byrepetitive iterations of the following steps: a) removal of theprotecting group from the 5′-hydroxyl group of the 3′-solidsupport-bound nucleoside or nucleic acid, b) coupling of the activatednucleoside phosphoramidite to the 5′-hydroxyl group, c) oxidation of thephosphite triester to the phosphate triester, and d) capping ofunreacted 5′-hydroxyl groups. DNA or RNA containing phosphorothioatelinkages is prepared as described above, except that the oxidation stepis replaced with a sulfurization step. Once the desired oligonucleotidesequence has been synthesized, the oligonucleotide is removed from thesupport, the phosphate triester groups are deprotected to phosphatediesters and the nucleoside bases are deprotected using aqueous ammoniaor other bases. See, for example, Beaucage (1993)“Oligodeoxyribonucleotide Synthesis” in PROTOCOLS FOR OLIGONUCLEOTIDESAND ANALOGS, SYNTHESIS AND PROPERTIES (Agrawal, ed.) Humana Press,Totowa, N.J.; Warner et al. (1984) DNA 3:401; Tang et al. (2000) Org.Process Res. Dev. 4:194-198; Wyrzykiewica et al. (1994) Bioorg. & Med.Chem. Lett. 4:1519-1522; Radhakrishna et al. (1989) J. Org. Chem.55:4693-4699. and U.S. Pat. No. 4,458,066. Programmable machines thatautomatically synthesize nucleic acid moieties of specified sequencesare widely available. Examples include the Expedite 8909 automated DNAsynthesizer (Perseptive Biosystem, Framington Mass.); the ABI 394(Applied Biosystems, Inc., Foster City, Calif.); and the OligoPilot II(Amersham Pharmacia Biotech, Piscataway, N.J.)

Polynucleotides can be assembled in the 3′ to 5′ direction, e.g., usingbase-protected nucleosides (monomers) containing an acid-labile5′-protecting group and a 3′-phosphoramidite. Examples of such monomersinclude 5′-O-(4,4′-dimethoxytrityl)-protectednucleoside-3′-O—(N,N-diisopropylamino) 2-cyanoethyl phosphoramidite,where examples of the protected nucleosides include, but are not limitedto, N6-benzoyladenosine, N4-benzoylcytidine, N2-isobutryrylguanosine,thymidine, and uridine. In this case, the solid support used contains a3′-linked protected nucleoside. Alternatively, polynucleotides can beassembled in the 5′ to 3′ direction using base-protected nucleosidescontaining an acid-labile 3′-protecting group and a 5′-phosphoramidite.Examples of such monomers include 3′-O-(4,4′-dimethoxytrityl)-protectednucleoside-5′-O—(N,N-diisopropylamino) 2-cyanoethyl phosphoramidite,where examples of the protected nucleosides include, but are not limitedto, N6-benzoyladenosine, N4-benzoylcytidine, N2-isobutryrylguanosine,thymidine, and uridine (Glen Research, Sterling, Va.). In this case, thesolid support used contains a 5′-linked protected nucleoside. Circularnucleic acid components can be isolated, synthesized through recombinantmethods, or chemically synthesized. Chemical synthesis can be performedusing any method described in the literature. See, for instance, Gao etal. (1995) Nucleic Acids Res. 23:2025-2029 and Wang et al. (1994)Nucleic Acids Res. 22:2326-2333.

Conjugation of the nucleic acid moieties and spacer moieties can becarried out in a variety of ways, depending on the particular CIC beingprepared. Methods for addition of particular spacer moieties are knownin the art and, for example, are described in the references citedsupra. See, e.g., Durand et al., Nucleic Acids Research 18:6353-59(1990). The covalent linkage between a spacer moiety and nucleic acidmoiety can be any of a number of types, including phosphodiester,phosphorothioate, amide, ester, ether, thioether, disulfide,phosphoramidate, phosphotriester, phosphorodithioate, methyl phosphonateand other linkages. As noted supra, spacer moiety precursors canoptionally be modified with terminal activating groups for coupling tonucleic acids. Examples of activated spacer moieties can be seen in FIG.1 where protecting groups suitable for automated synthesis have beenadded. Other spacer moiety precursors include, for example and not forlimitation, (1) HOCH₂CH₂—O—(CH₂CH₂O)_(n)CH₂CH₂OH, where n=0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44 or 45 or is greater than 45; (2) HOCH₂CHOHCH₂OH; (3)HO(CH₂)_(n)OH, where n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

In one embodiment, a spacer moiety precursor is used that includes firstand second reactive groups to permit conjugation to nucleic acidmoieties in a stepwise fashion, in which the first reactive group hasthe property that it can couple efficiently to the terminus of a growingchain of nucleic acids and the second reactive group is capable offurther extending, in a step-wise fashion the growing chain of mixednucleotide and non-nucleotide moieties in the CIC. It will often beconvenient to combine a spacer moiety(s) and a nucleic acid moiety(s)using the same phosphoramidite-type chemistry used for synthesis of thenucleic acid moiety. For example, CICs of the invention can beconveniently synthesized using an automated DNA synthesizer (e.g.,Expedite 8909; Perseptive Biosystems, Framington, Mass.) usingphosphoramidite chemistry (see, e.g., Beaucage, 1993, supra; CurrentProtocols in Nucleic Acid Chemistry, supra). However, one of skill willunderstand that the same (or equivalent) synthesis steps carried out byan automated DNA synthesizer can also be carried out manually, ifdesired. The resulting linkage between the nucleic acid and the spacerprecursors can be a phosphorothioate or phosphodiester linkage. In sucha synthesis, typically, one end of the spacer (or spacer subunit formultimeric spacers) is protected with a 4,4′-dimethoxytrityl group,while the other end contains a phosphoramidite group.

A variety of spacers with useful protecting and reacting groups arecommercially available, for example:

triethylene glycol spacer or “TEG spacer”9-O-(4,4′-dimethoxytrityl)triethyleneglycol-1-O-[(2-cyanoethyl)N,N-diisopropylphosphoramidite] (Glen Research, 22825 Davis Drive,Sterling, Va.);

hexaethylene glycol spacer or “HEG spacer”18-O-(4,4′-dimethoxytrityl)hexaethyleneglycol-1-O-[(2-cyanoethyl)N,N-diisopropylphosphoramidite] (Glen Research, Sterling, Va.);

propyl spacer 3-(4,4′-dimethoxytrityloxy)propyloxy-1-O-[(2-cyanoethyl)N,N-diisopropylphosphoramidite] (Glen Research, Sterling, Va.);

butyl spacer 4-(4,4′-dimethoxytrityloxy)butyloxy-1-O-[(2-cyanoethyl)N,N-diisopropylphosphoramidite] (Chem Genes Corporation, AshlandTechnology Center, 200 Horner Ave, Ashland, Mass.);

Hexyl spacer: 6-(4,4′-dimethoxytrityloxy)hexyloxy-1-O-[(2-cyanoethyl)N,N-diisopropylphosphoramidite] (Biosearch Technologies, Novoto, Calif.)

2-(hydroxymethyl)ethyl spacer or “HME spacer”1-(4,4′-dimethoxytrityloxy)-3-(levulinyloxy)-propyloxy-2-O-[(2-cyanoethyl)N,N-diisopropylphosphoramidite]; also called “asymmetrical branched”spacer (see FIG. 2) (Chem Genes Corp., Ashland Technology Center,Ashland Mass.);

“abasic nucleotide spacer” or “abasic spacer”5-O-(4,4′-dimethoxytrityl)-1,2-dideoxyribose-3-O-[(2-cyanoethyl)N,N-diisopropylphosphoramidite] (Glen Research, Sterling, Va.);

“symmetrical branched spacer” or “glycerol spacer”1,3-O,O-bis(4,4′-dimethoxytrityl)glycerol-2-O-[(2-cyanoethyl)N,N-diisopropylphosphoramidite] (Chem Genes, Ashland, Mass.) (see FIG.2);

“trebler spacer” (see FIG. 2)2,2,2-O,O,O-tris[3-O-(4,4′-dimethoxytrityloxy)propyloxymethyl]ethyl-1-O-[(2-cyanoethyl)N,N-diisopropylphosphoramidite] (Glen Research, Sterling, Va.);

“symmetrical doubler spacer” (see FIG. 2)1,3-O,O-bis[5-O-(4,4′-dimethoxytrityloxy)pentylamido]propyl-2-O-[(2-cyanoethyl)N,N-diisopropylphosphoramidite] (Glen Research, Sterling, Va.);

“dodecyl spacer”12-(4,4′-dimethoxytrityloxy)dodecyloxy-1-O-[(2-cyanoethyl)N,N-diisopropylphosphoramidite] (Glen Research, Sterling, Va.).

These and a large variety of other protected spacer moiety precursors(e.g., comprising DMT and phosphoramidite group protecting groups) canbe purchased or can be synthesized using routine methods for use inpreparing CICs disclosed herein. The instrument is programmed accordingto the manufacturer's instructions to add nucleotide monomers andspacers in the desired order.

CICs prepared “in situ” on a DNA synthesizer require protectednucleoside and protected spacer monomers, both containing reactive oractivatable functional groups. The reactive and/or protected form of thespacer moiety can be described as a “spacer precursor component.” Itwill be appreciated by those with skill in the art that the reactivegroups in the spacer precursors form stable linkages after coupling andthe protecting groups on the spacer precursor are removed in theresultant spacer moiety in the CIC. The protecting groups are generallyremoved during the step-wise synthesis of the CIC, in order to allowreaction at that site. In cases where there are protecting groups onadditional reactive groups, the protecting groups are removed after thestep-wise synthesis of the CIC (such as the levulinyl group on thespacer precursor shown in FIG. 2, structure 3, used to make C-25).

An example of a spacer precursor with no additional reactivefunctionality is18-O-(4,4′-dimethoxytrityl)hexaethyleneglycol-1-O-[(2-cyanoethyl)N,N-diisopropylphosphoramidite],which contains a protecting group, the 4,4′-dimethoxytrityl group, and areactive group, the (2-cyanoethyl)N,N-diisopropylphosphoramidite group.During preparation of the CIC using phosphoramidite chemistry on a DNAsynthesizer, the (2-cyanoethyl)N,N-diisopropylphosphoramidite group inthe spacer precursor is activated by a weak acid, such as 1H-tetrazole,and reacted with the free 5′-hydroxyl of the nucleobase-protectednucleic acid moiety to form a phosphite triester. The phosphite triestergroup is then either oxidized or sulfurized to a stable phosphotriesteror phosphorothioate triester group, respectively. The resultant triestergroup is stable to the rest of the CIC synthesis and remains in thatform until the final deprotection. In order to couple either anotherspacer precursor or an activated nucleoside monomer, which will becomepart of the next nucleic acid moiety, the 4,4′-dimethoxytrityl group onthe spacer precursor is removed. After coupling and either oxidation orsulfurization, this group also becomes either a stable phosphotriesteror phosphorothioate triester group, respectively. Once the protected CICis fully assembled, the CIC is cleaved from the solid support, thecyanoethyl groups on the phosphotriester or phosphorothioate triestergroups are removed, and the nucleobase protection is removed. In thisexample, the CIC contains spacer moieties including stablephosphodiester or phosphorothioate diester linkages to the nucleic acidmoieties. Both the reactive phosphoramidite group and the protectedhydroxyl group of the spacer precursor are converted to stablephosphodiester or phosphorothioate diester linkages in the spacermoiety. Because the reaction of each end of the spacer may beindependent, one linkage may be phosphodiester and the other linkagephosphorothioate diester, or any combination thereof. CICs with otherphosphate modifications, such as phosphorodithioate, methyl phosphonate,and phosphoramidate, may also be prepared in this manner by using aspacer precursor with the appropriate reactive group, the correctancillary reagents, and protocols designed for that type of linkage.These protocols are analogous to those described for preparing nucleicacid moieties with modified phosphate linkages.

Although use of phosphoramidite chemistry is convenient for thepreparation of certain CICs, it will be appreciated that the CICs of theinvention are not limited to compounds prepared by any particular methodof synthesis or preparation. For example, nucleic acid moietiescontaining groups not compatible with DNA synthesis and deprotectionconditions, such as (but not limited to) hydrazine or maleimide, can beprepared by reacting a nucleic acid moiety containing an amino linkerwith the appropriate heterobifunctional crosslinking reagent, such asSHNH (succinimidyl hydraziniumnicotinate) or sulfo-SMCC(sulfosuccinimidyl 4-[N-maleimidomethyl]-cyclohexame-1-carboxylate).

Methods for conjugating protein, peptides, oligonucleotides, and smallmolecules in various combinations are described in the literature andcan be adapted to achieve conjugation of a nucleic acid moietycontaining a reactive linking group to a spacer moiety precursor. See,for example, Bioconjugate Techniques, Greg T. Hermanson, Academic Press,Inc., San Diego, Calif., 1996. In some embodiments, a nucleic acidmoiety(s) is synthesized, and a reactive linking group (e.g., amino,carboxylate, thio, disulfide, and the like) is added using standardsynthetic chemistry techniques. The reactive linking group (which isconsidered to form a portion of the resulting spacer moiety) isconjugated to additional non-nucleic acid compounds (for example,without limitation, a compound listed in §4, supra) to form a portion ofthe spacer moiety. Reactive linking groups are added to nucleic acidsusing standard methods for nucleic acid synthesis, employing a varietyof reagents known in the art. Examples include reagents that contain aprotected amino group, carboxylate group, thiol group, disulfide group,aldehyde group, diol group, diene group and a phosphoramidite group.Once these compounds are incorporated into the nucleic acids, via theactivated phosphoramidite group, and are deprotected, they providenucleic acids with amino, carboxylate, aldehyde, diol, diene or thiolreactivity. Examples of reactive groups for conjugating a nucleic acidmoiety containing a reactive linker group to a spacer moiety precursorthat contains a reactive group are shown below.

nucleic acid reactive Spacer moiety precursor group reactive groupStable linkage formed thiol maleimide, haloacetyl thioether maleimidethiol thioether thiol pyridine disulfide disulfide pyridine disulfidethiol disulfide amine NHS or other active ester amide amine carboxylateamide carboxylate amine amide aldehyde, ketone hydrazine, hydrazidehydrazone, hydrazide hydrazine, hydrazide aldehyde, ketone hydrazone,hydrazide diene dienophile aliphatic or heterocyclic ring

The reactive linking group and the spacer precursor react to form astable bond and the entire group of atoms between the two (or more)nucleic acid moieties is defined as the spacer moiety. For example, anucleic acid moiety synthesized with a mercaptohexyl group linked to thenucleic acid moiety via a phosphorothioate group can be reacted with aspacer precursor containing one (or more) maleimide group(s), forming athioether linkage(s). The spacer moiety of this CIC includes thephosphorothioate group and hexyl group from the linker on the nucleicacid moiety, the new thioether linkage, and the rest of the spacer thatwas part of the spacer precursor.

Although linear CICs can be made using these conjugation strategies,these methods are most often applied for the preparation of branchedCICs. Additionally, spacer precursor molecules can be prepared withseveral orthogonal reactive groups to allow for the addition of morethan one type nucleic acid moiety (e.g., different sequence motif).

In one embodiment, CICs with multivalent spacers conjugated to more thanone type of nucleic acid moiety are prepared. For instance, platformscontaining two maleimide groups (which can react with thiol-containingpolynucleotides), and two activated ester groups (which can react withamino-containing nucleic acids) have been described (see, e.g.,PCT/US94/10031, published as WO 95/07073). These two activated groupscan be reacted independently of each other. This would result in a CICcontaining a total of 4 nucleic acid moieties, two of each sequence.

CICs with multivalent spacers containing two different nucleic acidsequences can also be prepared using the symmetrical branched spacer,described above, and conventional phosphoramidite chemistry (e.g., usingmanual or automated methods). The symmetrical branched spacer contains aphosphoramidite group and two protecting groups that are the same andare removed simultaneously. In one approach, for example, a firstnucleic acid is synthesized and coupled to the symmetrical branchedspacer, the protecting groups are removed from the spacer. Then twoadditional nucleic acids (of the same sequence) are synthesized on thespacer (using double the amount of reagents used for synthesis of asingle nucleic acid moiety in each step). This procedure is described indetail in Example 15, infra.

A similar method can be used to connect three different nucleic acidmoieties (referred to below as Nucleic acids I, II, and III) to amultivalent platform (e.g., asymmetrical branched spacer). This is mostconveniently carried out using an automated DNA synthesizer. In oneembodiment, the asymmetrical branched spacer contains a phosphoramiditegroup and two orthogonal protecting groups that can be removedindependently. First, nucleic acid I is synthesized, then theasymmetrical branched spacer is coupled to nucleic acid I, then nucleicacid II is added after the selective removal of one of the protectinggroups. Nucleic acid II is deprotected, and capped, and then the otherprotecting group on the spacer is removed. Finally, nucleic acid III issynthesized. This procedure is described in detail in Example 17, infra.

Hydrophilic linkers of variable lengths are may be used, for example tolink nucleic acids moieties and platform molecules. A variety ofsuitable linkers are known. Suitable linkers include, withoutlimitation, linear oligomers or polymers of ethylene glycol. Suchlinkers include linkers with the formulaR¹S(CH₂CH₂O)_(n)CH₂CH₂—O—(CH₂)_(m)CO₂R² wherein n=0-200, m=1 or 2, R¹=Hor a protecting group such as trityl, R²=H or alkyl or aryl, e.g.,4-nitrophenyl ester. These linkers may be used in connecting a moleculecontaining a thiol reactive group such as haloaceyl, maleiamide, etc.,via a thioether to a second molecule which contains an amino group viaan amide bond. The order of attachment can vary, i.e., the thioetherbond can be formed before or after the amide bond is formed. Otherlinkers include Sulfo-SMCC (sulfosuccinimidyl4-[N-maleimidomethyl]-cyclohexane-1-carboxylate) Pierce Chemical Co.product 22322; Sulfo-EMCS (N-[ε-maleimidocaproyloxyl sulfosuccinimideester) Pierce Chemical Co. product 22307; Sulfo-GMBS(N-[γ-maleimidobutyryloxy]sulfosuccinimide ester) Pierce Chemical Co.product 22324 (Pierce Chemical Company, Rockford, Ill.), and similarcompounds of the general formula maleimido-R—C(O)NHS ester, whereR=alkyl, cyclic alkyl, polymers of ethylene glycol, and the like.

6. Proteinaceous CICs

In certain embodiments, a polypeptide, such as a protein antigen orantigen fragment, is used as a multivalent spacer moiety to which aplurality of nucleic acid moieties are covalently conjugated, directlyor via linkers, to form a “proteinaceous CIC.” The polypeptide can be anantigen or immunogen to which an adaptive immune response is desired, ora carrier (e.g., albumin). Typically, a proteinaceous CIC comprises atleast one, and usually several or many nucleic acid moieties that (a)are between 2 and 7, more often between 4 and 7 nucleotides in length,alternatively between 2 and 6, 2 and 5, 4 and 6, or 4 and 5 nucleotidesin length and/or (b) have inferior isolated immunomodulatory activity ordo not have isolated immunomodulatory activity. Methods of making aproteinaceous CIC will be apparent to one of skill upon review of thepresent disclosure. A nucleic acid, for example, can be covalentlyconjugated to a polypeptide spacer moiety by art known methods includinglinkages between a 3′ or 5′ end of a nucleic acid moiety (or at asuitably modified base at an internal position in the a nucleic acidmoiety) and a polypeptide with a suitable reactive group (e.g., anN-hydroxysuccinimide ester, which can be reacted directly with the N⁴amino group of cytosine residues). As a further example, a polypeptidecan be attached to a free 5′-end of a nucleic acid moiety through anamine, thiol, or carboxyl group that has been incorporated into nucleicacid moiety. Alternatively, the polypeptide can be conjugated to aspacer moiety, as described herein. Further, a linking group comprisinga protected amine, thiol, or carboxyl at one end, and a phosphoramiditecan be covalently attached to a hydroxyl group of a polynucleotide, and,subsequent to deprotection, the functionality can be used to covalentlyattach the CIC to a peptide.

7. Purification

The CICs of the invention are purified using any conventional means,such as high performance liquid chromatography, electrophoretic methods,nucleic acid affinity chromatography, size exclusion chromatography, andion exchange chromatography. In some embodiments, a CIC is substantiallypure, e.g., at least about 80% pure by weight, often at least about 90%pure by weight, more often at least about 95% pure, most often at leastabout 85% pure.

8. Compositions

In various embodiments, compositions of the invention comprise one ormore CICs, (i.e. a single CIC or a combination of two or more CICs)optionally in conjunction with another immunomodulatory agent, such as apeptide, an antigen (described below) and/or an additional adjuvant.Compositions of the invention may comprise a CIC and pharmaceuticallyacceptable excipient. By “pharmaceutically acceptable” it is meant thecarrier, diluent or excipient must be compatible with the otheringredients of the formulation and not deleterious to the recipientthereof. Pharmaceutically acceptable excipients are well known in theart and include sterile water, isotonic solutions such as saline andphosphate buffered saline, and other excipients known in the art. See,e.g., Remington: The Science and Practice of Pharmacy (19th edition,1995, Gennavo, ed.). Adjuvants (an example of which is alum) are knownin the art. CIC formulations may be prepared with otherimmunotherapeutic agents, such as cytokines and antibodies. In someembodiments the composition is isotonic and/or sterile, e.g., suitablefor administration to a human patient, e.g., manufactured or formulatedunder GMP standards.

A. CIC/MC Complexes

CICs may be administered in the form of CIC/microcarrier (CIC/MC)complexes. Accordingly, the invention provides compositions comprisingCIC/MC complexes.

CIC/MC complexes comprise a CIC bound to the surface of a microcarrier(i.e., the CIC is not encapsulated in the MC), and preferably comprisemultiple molecules of CIC bound to each microcarrier. In certainembodiments, a mixture of different CICs may be complexed with amicrocarrier, such that the microcarrier is bound to more than one CICspecies. The bond between the CIC and MC may be covalent or non-covalent(e.g. mediated by ionic and/or hydrophobic interactions). As will beunderstood by one of skill in the art, the CIC may be modified orderivatized and the composition of the microcarrier may be selectedand/or modified to accommodate the desired type of binding desired forCIC/MC complex formation.

Covalently bonded CIC/MC complexes may be linked using any covalentcrosslinking technology known in the art. Typically, the CIC portionwill be modified, either to incorporate an additional moiety (e.g., afree amine, carboxyl or sulfhydryl group) or incorporate modified (e.g.,phosphorothioate) nucleotide bases to provide a site at which the CICportion may be linked to the microcarrier. The link between the CIC andMC portions of the complex can be made at the 3′ or 5′ end of the CIC,or at a suitably modified base at an internal position in the CIC. Themicrocarrier is generally also modified to incorporate moieties throughwhich a covalent link may be formed, although functional groups normallypresent on the microcarrier may also be utilized. The CIC/MC is formedby incubating the CIC with a microcarrier under conditions which permitthe formation of a covalent complex (e.g., in the presence of acrosslinking agent or by use of an activated microcarrier comprising anactivated moiety which will form a covalent bond with the CIC).

A wide variety of crosslinking technologies are known in the art, andinclude crosslinkers reactive with amino, carboxyl and sulfhydrylgroups. As will be apparent to one of skill in the art, the selection ofa crosslinking agent and crosslinking protocol will depend on theconfiguration of the CIC and the microcarrier as well as the desiredfinal configuration of the CIC/MC complex. The crosslinker may be eitherhomobifunctional or heterobifunctional. When a homobifunctionalcrosslinker is used, the crosslinker exploits the same moiety on the CICand MC (e.g., an aldehyde crosslinker may be used to covalently link aCIC and MC where both the CIC and MC comprise one or more free amines).Heterobifunctional crosslinkers utilize different moieties on the CICand MC, (e.g., a maleimido-N-hydroxysuccinimide ester may be used tocovalently link a free sulfhydryl on the CIC and a free amine on theMC), and are preferred to minimize formation of inter-microcarrierbonds. In most cases, it is preferable to crosslink through a firstcrosslinking moiety on the microcarrier and a second crosslinking moietyon the CIC, where the second crosslinking moiety is not present on themicrocarrier. One preferred method of producing the CIC/MC complex is by‘activating’ the microcarrier by incubating with a heterobifunctionalcrosslinking agent, then forming the CIC/MC complex by incubating theCIC and activated MC under conditions appropriate for reaction. Thecrosslinker may incorporate a “spacer” arm between the reactivemoieties, or the two reactive moieties in the crosslinker may bedirectly linked.

In one preferred embodiment, the CIC portion comprises at least one freesulfhydryl (e.g., provided by a 5′-thiol modified base or linker) forcrosslinking to the microcarrier, while the microcarrier comprises freeamine groups. A heterobifunctional crosslinker reactive with these twogroups (e.g., a crosslinker comprising a maleimide group and aNHS-ester), such as succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate is used to activate theMC, then covalently crosslink the CIC to form the CIC/MC complex.

Non-covalent CIC/MC complexes may be linked by any non-covalent bindingor interaction, including ionic (electrostatic) bonds, hydrophobicinteractions, hydrogen bonds, van der Waals attractions, or acombination of two or more different interactions, as is normally thecase when a binding pair is to link the CIC and MC.

Preferred non-covalent CIC/MC complexes are typically complexed byhydrophobic or electrostatic (ionic) interactions, or a combinationthereof, (e.g., through base pairing between a CIC and a polynucleotidebound to an MC). Due to the hydrophilic nature of the backbone ofpolynucleotides, CIC/MC complexes which rely on hydrophobic interactionsto form the complex generally require modification of the CIC portion ofthe complex to incorporate a highly hydrophobic moiety. Preferably, thehydrophobic moiety is biocompatible, nonimmunogenic, and is naturallyoccurring in the individual for whom the composition is intended (e.g.,is found in mammals, particularly humans). Examples of preferredhydrophobic moieties include lipids, steroids, sterols such ascholesterol, and terpenes. The method of linking the hydrophobic moietyto the CIC will, of course, depend on the configuration of the CIC andthe identity of the hydrophobic moiety. The hydrophobic moiety may beadded at any convenient site in the CIC, preferably at either the 5′ or3′ end; in the case of addition of a cholesterol moiety to a CIC, thecholesterol moiety is preferably added to the 5′ end of the CIC, usingconventional chemical reactions (see, for example, Godard et al. (1995)Eur. J. Biochem. 232:404-410). Preferably, microcarriers for use inCIC/MC complexes linked by hydrophobic bonding are made from hydrophobicmaterials, such as oil droplets or hydrophobic polymers, althoughhydrophilic materials modified to incorporate hydrophobic moieties maybe utilized as well. When the microcarrier is a liposome or other liquidphase microcarrier comprising a lumen, the CIC/MC complex is formed bymixing the CIC and the MC after preparation of the MC, in order to avoidencapsulation of the CIC during the MC preparation process.

Non-covalent CIC/MC complexes bound by electrostatic binding typicallyexploit the highly negative charge of the polynucleotide backbone.Accordingly, microcarriers for use in non-covalently bound CIC/MCcomplexes are generally positively charged at physiological pH (e.g.,about pH 6.8-7.4). The microcarrier may intrinsically possess a positivecharge, but microcarriers made from compounds not normally possessing apositive charge may be derivatized or otherwise modified to becomepositively charged. For example, the polymer used to make themicrocarrier may be derivatized to add positively charged groups, suchas primary amines. Alternately, positively charged compounds may beincorporated in the formulation of the microcarrier during manufacture(e.g., positively charged surfactants may be used during the manufactureof poly(lactic acid)/poly(glycolic acid) copolymers to confer a positivecharge on the resulting microcarrier particles). See, e.g., Examples 28and 34, infra.

Non-covalent CIC/MC complexes linked by nucleotide base pairing may beproduced using conventional methodologies. Generally, base-paired CIC/MCcomplexes are produced using a microcarrier comprising a bound,preferably a covalently bound, polynucleotide (the “capturepolynucleotide”) that is at least partially complementary to the CIC.The segment of complementarity between the CIC and the capturenucleotide is preferably at least 6, 8, 10 or 15 contiguous base pairs,more preferably at least 20 contiguous base pairs. The capturenucleotide may be bound to the MC by any method known in the art, and ispreferably covalently bound to the CIC at the 5′ or 3′ end.

In other embodiments, a binding pair may be used to link the CIC and MCin a CIC/MC complex. The binding pair may be a receptor and ligand, anantibody and antigen (or epitope), or any other binding pair which bindsat high affinity (e.g., K_(d) less than about 10⁻⁸). One type ofpreferred binding pair is biotin and streptavidin or biotin and avidin,which form very tight complexes. When using a binding pair to mediateCIC/MC complex binding, the CIC is derivatized, typically by a covalentlinkage, with one member of the binding pair, and the MC is derivatizedwith the other member of the binding pair. Mixture of the twoderivatized compounds results in CIC/MC complex formation.

Many CIC/MC complex embodiments do not include an antigen, and certainembodiments exclude antigen(s) associated with the disease or disorderwhich is the object of the CIC/MC complex therapy. In furtherembodiments, the CIC is also bound to one or more antigen molecules.Antigen may be coupled with the CIC portion of a CIC/MC complex in avariety of ways, including covalent and/or non-covalent interactions.Alternately, the antigen may be linked to the microcarrier. The linkbetween the antigen and the CIC in CIC/MC complexes comprising anantigen bound to the CIC can be made by techniques described herein andknown in the art.

B. Co-Administered Antigen

In some embodiments, the CIC is coadministered with an antigen. Anyantigen may be co-administered with a CIC and/or used for preparation ofcompositions comprising a CIC and antigen.

In some embodiments, the antigen is an allergen. Examples of recombinantallergens are provided in Table 1. Preparation of many allergens iswell-known in the art, including, but not limited to, preparation ofragweed pollen allergen Antigen E (Amb aI) (Rafnar et al. (1991) J.Biol. Chem. 266:1229-1236), grass allergen Lol p 1 (Tamborini et al.(1997) Eur. J. Biochem. 249:886-894), major dust mite allergens Der pIand Der PII (Chua et al. (1988) J. Exp. Med. 167:175-182; Chua et al.(1990) Int. Arch. Allergy Appl. Immunol. 91:124-129), domestic catallergen Fel d I (Rogers et al. (1993) Mol. Immunol. 30:559-568), whitebirch pollen Bet vl (Breiteneder et al. (1989) EMBO J. 8:1935-1938),Japanese cedar allergens Cry j 1 and Cry j 2 (Kingetsu et al. (2000)Immunology 99:625-629), and protein antigens from other tree pollen(Elsayed et al. (1991) Scand. J. Clin. Lab. Invest. Suppl. 204:17-31).Preparation of protein antigens from grass pollen for in vivoadministration has been reported.

In some embodiments, the allergen is a food allergen, including, but notlimited to, peanut allergen, for example Ara h I (Stanley et al. (1996)Adv. Exp. Med. Biol. 409:213-216); walnut allergen, for example, Jug r I(Tueber et al. (1998) J. Allergy Clin. Immunol. 101:807-814); brazil nutallergen, for example, albumin (Pastorello et al. (1998) J. AllergyClin: Immunol. 102:1021-1027; shrimp allergen, for example, Pen a I(Reese et al. (1997) Int. Arch. Allergy Immunol. 113:240-242); eggallergen, for example, ovomucoid (Crooke et al. (1997) J. Immunol.159:2026-2032); milk allergen, for example, bovine β-lactoglobin (Selotal. (1999) Clin. Exp. Allergy 29:1055-1063); fish allergen, for example,parvalbumins (Van Do et al. (1999) Scand. J. Immunol. 50:619-625;Galland et al. (1998) J. Chromatogr. B. Biomed. Sci. Appl. 706:63-71).In some embodiments, the allergen is a latex allergen, including but notlimited to, Hey b 7 (Sowka et al. (1998) Eur. J. Biochem. 255:213-219).Table 1 shows a list of allergens that may be used.

TABLE 1 RECOMBINANT ALLERGENS Group Allergen Reference ANIMALS:CRUSTACEA Shrimp/lobster tropomyosin Leung et al. (1996) J. AllergyClin. Immunol. 98: 954-961 Pan s I Leung et al. (1998) Mol. Mar. Biol.Biotechnol. 7: 12-20 INSECTS Ant Sol i 2 (venom) Schmidt et al. JAllergy Clin Immunol., 1996, 98: 82-8 Bee Phospholipase A2 (PLA) Mulleret al. J Allergy Clin Immunol, 1995, 96: 395-402 Forster et al. JAllergy Clin Immunol, 1995, 95: 1229-35 Muller et al. Clin Exp Allergy,1997, 27: 915-20 Hyaluronidase (Hya) Soldatova et al. J Allergy ClinImmunol, 1998, 101: 691-8 Cockroach Bla g Bd9OK Helm et al. J AllergyClin Immunol, 1996, 98: 172-180 Bla g 4 (a calycin) Vailes et al. JAllergy Clin Immunol, 1998, 101: 274-280 Glutathione S-transferaseArruda et al. J Biol Chem, 1997, 272: 20907-12 Per a 3 Wu et al. MolImmunol, 1997, 34: 1-8 Dust mite Der p 2 (major allergen) Lynch et al. JAllergy Clin Immunol, 1998, 101: 562-4 Hakkaart et al. Clin Exp Allergy,1998, 28: 169-74 Hakkaart et al. Clin Exp Allergy, 1998, 28: 45-52Hakkaart et al. Int Arch Allergy Immunol, 1998, 115 (2): 150-6 Muelleret al. J Biol Chem, 1997, 272: 26893-8 Der p2 variant Smith et al. JAllergy Clin Immunol, 1998, 101: 423-5 Der f2 Yasue et al. Clin ExpImmunol, 1998, 113: 1-9 Yasue et al. Cell Immunol, 1997, 181: 30-7 Derp10 Asturias et al. Biochim Biophys Acta, 1998, 1397: 27-30 Tyr p 2Eriksson et al. Eur J Biochem, 1998 Hornet Antigen 5 aka Dol m VTomalski et al. Arch Insect Biochem Physiol, 1993, 22: 303-13 (venom)Mosquito Aed a I (salivary apyrase) Xu et al. Int Arch Allergy Immunol,1998, 115: 245-51 Yellow jacket antigen 5, hyaluronidase and King et al.J Allergy Clin Immunol, 1996, 98: 588-600 phospholipase (venom) MAMMALSCat Fel d I Slunt et al. J Allergy Clin Immunol, 1995, 95: 1221-8Hoffmann et al. (1997) J Allergy Clin Immunol 99: 227-32 Hedlin CurrOpin Pediatr, 1995, 7: 676-82 Cow Bos d 2 (dander; a lipocalin) Zeileret al. J Allergy Clin Immunol, 1997, 100: 721-7 Rautiainen et al.Biochem Bioph. Res Comm., 1998, 247: 746-50 β-lactoglobulin (BLG, majorChatel et al. Mol Immunol, 1996, 33: 1113-8 cow milk allergen) Lehrer etal. Crit Rev Food Sci Nutr, 1996, 36: 553-64 Dog Can f I and Can f 2,salivary Konieczny et al. Immunology, 1997, 92: 577-86 lipocalinsSpitzauer et al. J Allergy Clin Immunol, 1994, 93: 614-27 Vrtala et al.J Immunol, 1998, 160: 6137-44 Horse Equ c1 (major allergen, a Gregoireet al. J Biol Chem, 1996, 271: 32951-9 lipocalin) Mouse mouse urinaryprotein (MUP) Konieczny et al. Immunology, 1997, 92: 577-86 OTHERMAMMALIAN ALLERGENS Insulin Ganz et al. J Allergy Clin Immunol, 1990,86: 45-51 Grammer et al. J Lab Clin Med, 1987, 109: 141-6 Gonzalo et al.Allergy, 1998, 53: 106-7 Interferons interferon alpha 2c Detmar et al.Contact Dermatis, 1989, 20: 149-50 MOLLUSCS topomyosin Leung et al. JAllergy Clin Immunol, 1996, 98: 954-61 PLANT ALLERGENS: Barley Hor v 9Astwood et al. Adv Exp Med Biol, 1996, 409: 269-77 Birch pollenallergen, Bet v 4 Twardosz et al. Biochem Bioph. Res Comm., 1997, 23 9:197 rBet v 1 Bet v 2: (profilin) Pauli et al. J Allergy Clin Immunol,1996, 97: 1100-9 van Neerven et al. Clin Exp Allergy, 1998, 28: 423-33Jahn-Schmid et al. Immunotechnology, 1996, 2: 103-13 Breitwieser et al.Biotechniques, 1996, 21: 918-25 Fuchs et al. J Allergy Clin Immunol,1997, 100: 356-64 Brazil nut globulin Bartolome et al. AllergolImmunopathol, 1997, 25: 135-44 Cherry Pru a I (major allergen) Scheureret al. Mol Immunol, 1997, 34: 619-29 Corn Zml3 (pollen) Heiss et al.FEBS Lett, 1996, 381: 217-21 Lehrer et al. Int Arch Allergy Immunol,1997, 113: 122-4 Grass Phl p 1, Phl p 2, Phl p 5 Bufe et al. Am J RespirCrit Care Med, 1998, 157: 1269-76 (timothy grass pollen) Vrtala et al. JImmunol Jun. 15, 1998, 160: 6137-44 Niederberger et al. J Allergy ClinImmun., 1998, 101: 258-64 Hol l 5 velvet grass pollen Schramm et al. EurJ Biochem, 1998, 252: 200-6 Bluegrass allergen Zhang et al. J Immunol,1993, 151: 791-9 Cyn d 7 Bermuda grass Smith et al. Int Arch AllergyImmunol, 1997, 114: 265-71 Cyn d 12 (a profilin) Asturias et al. ClinExp Allergy, 1997, 27: 1307-13 Fuchs et al. J Allergy Clin Immunol,1997, 100: 356-64 Japanese Cedar Jun a 2 (Juniperus ashei) Yokoyama etal. Biochem. Biophys. Res. Commun., 2000, 275: 195-202 Cry j 1, Cry j 2(Cryptomeria Kingetsu et al. Immunology, 2000, 99: 625-629 japonica)Juniper Jun o 2 (pollen) Tinghino et al. J Allergy Clin Immunol, 1998,101: 772-7 Latex Hev b 7 Sowka et al. Eur J Biochem, 1998, 255: 213-9Fuchs et al. J Allergy Clin Immunol, 1997, 100: 356-64 Mercurialis Mer aI (profilin) Vallverdu et al. J Allergy Clin Immunol, 1998, 101: 363-70Mustard (Yellow) Sin a I (seed) Gonzalez de la Pena et al. BiochemBioph. Res Comm., 1993, 190: 648-53 Oilseed rape Bra r I pollen allergenSmith et al. Int Arch Allergy Immunol, 1997, 114: 265-71 Peanut Ara h IStanley et al. Adv Exp Med Biol, 1996, 409: 213-6 Burks et al. J ClinInvest, 1995, 96: 1715-21 Burks et al. Int Arch Allergy Immunol, 1995,107: 248-50 Poa pratensis Poa p9 Parronchi et al. Eur J Immunol, 1996,26: 697-703 Astwood et al. Adv Exp Med Biol, 1996, 409: 269-77 RagweedAmb a I Sun et al. Biotechnology August, 1995, 13: 779-86 Hirschwehr etal. J Allergy Clin Immunol, 1998, 101: 196-206 Casale et al. J AllergyClin Immunol, 1997, 100: 110-21 Rye Lol p I Tamborini et al. Eur JBiochem, 1997, 249: 886-94 Walnut Jug r I Teuber et al. J Allergy ClinImmun., 1998, 101: 807-14 Wheat allergen Fuchs et al. J Allergy ClinImmunol, 1997, 100: 356-64 Donovan et al. Electrophoresis, 1993, 14:917-22 FUNGI: Aspergillus Asp f 1, Asp f 2, Asp f3, Asp f Crameri et al.Mycoses, 1998, 41 Suppl 1: 56-60 4, rAsp f 6 Hemmann et al. Eur JImmunol, 1998, 28: 1155-60 Banerjee et al. J Allergy Clin Immunol, 1997,99: 821-7 Crameri Int Arch Allergy Immunol, 1998, 115: 99-114 Crameri etal. Adv Exp Med Biol, 1996, 409: 111-6 Moser et al. J Allergy ClinImmunol, 1994, 93: 1-11 Manganese superoxide Mayer et al. Int ArchAllergy Immunol, 1997, 113: 213-5 dismutase (MNSOD) Blomia allergenCaraballo et al. Adv Exp Med Biol, 1996, 409: 81-3 Penicilliniumallergen Shen et al. Clin Exp Allergy, 1997, 27: 682-90 Psilocybe Psi c2 Horner et al. Int Arch Allergy Immunol, 1995, 107: 298-300

In some embodiments, the antigen is from an infectious agent, includingprotozoan, bacterial, fungal (including unicellular and multicellular),and viral infectious agents. Examples of suitable viral antigens aredescribed herein and are known in the art. Bacteria include Hemophilusinfluenza, Mycobacterium tuberculosis and Bordetella pertussis.Protozoan infectious agents include malarial plasmodia, Leishmaniaspecies, Trypanosoma species and Schistosoma species. Fungi includeCandida albicans.

In some embodiments, the antigen is a viral antigen. Viral polypeptideantigens include, but are not limited to, HIV proteins such as HIV gagproteins (including, but not limited to, membrane anchoring (MA)protein, core capsid (CA) protein and nucleocapsid (NC) protein), HIVpolymerase, influenza virus matrix (M) protein and influenza virusnucleocapsid (NP) protein, hepatitis B surface antigen (HBsAg),hepatitis B core protein (HBcAg), hepatitis e protein (HBeAg), hepatitisB DNA polymerase, hepatitis C antigens, and the like. Referencesdiscussing influenza vaccination include Scherle and Gerhard (1988)Proc. Natl. Acad. Sci. USA 85:4446-4450; Scherle and Gerhard (1986) J.Exp. Med. 164:1114-1128; Granoff et al. (1993) Vaccine 11:S46-51;Kodihalli et al. (1997) J. Virol. 71:3391-3396; Ahmeida et al. (1993)Vaccine 11:1302-1309; Chen et al. (1999) Vaccine 17:653-659; Govorkovaand Smirnov (1997) Acta Virol. (1997) 41:251-257; Koide et al. (1995)Vaccine 13:3-5; Mbawuike et al. (1994) Vaccine 12:1340-1348; Tamura etal. (1994) Vaccine 12:310-316; Tamura et al. (1992) Eur. J. Immunol.22:477-481; Hirabayashi et al. (1990) Vaccine 8:595-599. Other examplesof antigen polypeptides are group- or sub-group specific antigens, whichare known for a number of infectious agents, including, but not limitedto, adenovirus, herpes simplex virus, papilloma virus, respiratorysyncytial virus and poxviruses.

Many antigenic peptides and proteins are known, and available in theart;

others can be identified using conventional techniques. For immunizationagainst tumor formation or treatment of existing tumors,immunomodulatory peptides can include tumor cells (live or irradiated),tumor cell extracts, or protein subunits of tumor antigens such asHer-2/neu, Martl, carcinoembryonic antigen (CEA), gangliosides, humanmilk fat globule (HMFG), mucin (MUCl), MAGE antigens, BAGE antigens,GAGE antigens, gp100, prostate specific antigen (PSA), and tyrosinase.Vaccines for immuno-based contraception can be formed by including spermproteins administered with CICs. Lea et al. (1996) Biochim. Biophys.Acta 1307:263.

Attenuated and inactivated viruses are suitable for use herein as theantigen. Preparation of these viruses is well-known in the art and manyare commercially available (see, e.g., Physicians' Desk Reference (1998)52nd edition, Medical Economics Company, Inc.). For example, polio virusis available as IPOL® (Pasteur Merieux Connaught) and ORIMUNE® (LederleLaboratories), hepatitis A virus as VAQTA® (Merck), measles virus asATTENUVAX® (Merck), mumps virus as MUMPSVAX® (Merck) and rubella virusas MERUVAX®II (Merck). Additionally, attenuated and inactivated virusessuch as HIV-1, HIV-2, herpes simplex virus, hepatitis B virus,rotavirus, human and non-human papillomavirus and slow brain viruses canprovide peptide antigens.

In some embodiments, the antigen comprises a viral vector, such asvaccinia, adenovirus, and canary pox.

Antigens may be isolated from their source using purification techniquesknown in the art or, more conveniently, may be produced usingrecombinant methods.

Antigenic peptides can include purified native peptides, syntheticpeptides, recombinant proteins, crude protein extracts, attenuated orinactivated viruses, cells, micro-organisms, or fragments of suchpeptides. Immunomodulatory peptides can be native or synthesizedchemically or enzymatically. Any method of chemical synthesis known inthe art is suitable. Solution phase peptide synthesis can be used toconstruct peptides of moderate size or, for the chemical construction ofpeptides, solid phase synthesis can be employed. Atherton et al. (1981)Hoppe Seylers Z. Physiol. Chem. 362:833-839. Proteolytic enzymes canalso be utilized to couple amino acids to produce peptides. Kullmann(1987) Enzymatic Peptide Synthesis, CRC Press, Inc. Alternatively, thepeptide can be obtained by using the biochemical machinery of a cell, orby isolation from a biological source. Recombinant DNA techniques can beemployed for the production of peptides. Hames et al. (1987)Transcription and Translation: A Practical Approach, IRL Press. Peptidescan also be isolated using standard techniques such as affinitychromatography.

Preferably the antigens are peptides, lipids (e.g., sterols excludingcholesterol, fatty acids, and phospholipids), polysaccharides such asthose used in H. influenza vaccines, gangliosides and glycoproteins.These can be obtained through several methods known in the art,including isolation and synthesis using chemical and enzymatic methods.In certain cases, such as for many sterols, fatty acids andphospholipids, the antigenic portions of the molecules are commerciallyavailable.

Examples of viral antigens useful in the subject compositions andmethods using the compositions include, but are not limited to, HIVantigens. Such antigens include, but are not limited to, those antigensderived from HIV envelope glycoproteins including, but not limited to,gp160, gp120 and gp41. Numerous sequences for HIV genes and antigens areknown. For example, the Los Alamos National Laboratory HIV SequenceDatabase collects, curates and annotates HIV nucleotide and amino acidsequences. This database is accessible via the internet, athttp://hiv-web.1an1.gov/, and in a yearly publication, see HumanRetroviruses and AIDS Compendium (for example, 2000 edition).

Antigens derived from infectious agents may be obtained using methodsknown in the art, for example, from native viral or bacterial extracts,from cells infected with the infectious agent, from purifiedpolypeptides, from recombinantly produced polypeptides and/or assynthetic peptides.

CICs can be administered in combination with antigen in a variety ofways. In some embodiments, a CIC and antigen are administered spatiallyproximate with respect to each other. As described below, spatialproximation can be accomplished in a number of ways, includingconjugation, encapsidation, via affixation to a platform or adsorptiononto a surface. In one embodiment, a CIC and antigen are administered asan admixture (e.g., in solution). It is specifically contemplated that,in certain embodiments, the CIC is not conjugated to an immunogen orantigen.

In some embodiments, the CIC is linked to a polypeptide, e.g., anantigen. The CIC portion can be linked with the antigen portion of aconjugate in a variety of ways, including covalent and/or non-covalentinteractions, via the nucleic acid moiety or non-nucleic acid spacermoiety. In some embodiments, linkage is via a reactive group such as,without limitation, thio, amine, carboxylate, aldehyde, hydrizine,hydrizone, disulfide and the like.

The link between the portions can be made at the 3′ or 5′ end of anucleic acid moiety, or at a suitably modified base at an internalposition in the a nucleic acid moiety. For example, if the antigen is apeptide and contains a suitable reactive group (e.g., anN-hydroxysuccinimide ester) it can be reacted directly with the N⁴ aminogroup of cytosine residues. Depending on the number and location ofcytosine residues in the CIC, specific coupling at one or more residuescan be achieved.

Alternatively, modified oligonucleosides, such as are known in the art,can be incorporated at either terminus, or at internal positions in theCIC. These can contain blocked functional groups which, when deblocked,are reactive with a variety of functional groups which can be presenton, or attached to, the antigen of interest.

Where the antigen is a peptide, this portion of the conjugate can beattached to the nucleic acid moiety or spacer moiety through solidsupport chemistry. For example, a nucleic acid portion of a CIC can beadded to a polypeptide portion that has been pre-synthesized on asupport. Haralambidis et al. (1990) Nucleic Acids Res. 18:493-499; andHaralambidis et al. (1990) Nucleic Acids Res. 18:501-505.

Alternatively, the CIC can be synthesized such that it is connected to asolid support through a cleavable linker extending from the 3′-end of anucleic acid moiety. Upon chemical cleavage of the CIC from the support,a terminal thiol group or a terminal amino group is left at the 3′-endof the nucleic acid moiety (Zuckermann et al., 1987, Nucleic Acids Res.15:5305-5321; Corey et al., 1987, Science 238:1401-1403; Nelson et al.,1989, Nucleic Acids Res. 17:1781-1794). Conjugation of theamino-modified CIC to amino groups of the peptide can be performed asdescribed in Benoit et al. (1987) Neuromethods 6:43-72. Conjugation ofthe thiol-modified CIC to carboxyl groups of the peptide can beperformed as described in Sinah et al. (1991) Oligonucleotide Analogues:A Practical Approach, IRL Press. Coupling of a nucleic acid moiety orspacer carrying an appended maleimide to the thiol side chain of acysteine residue of a peptide has also been described. Tung et al.(1991) Bioconjug. Chem. 2:464-465.

The peptide portion of the conjugate can be attached to a free 5′-end ofa nucleic acid moiety through an amine, thiol, or carboxyl group thathas been incorporated into nucleic acid moiety or spacer (e.g., via afree 5′-end, a 3′-end, via a modified base, and the like).

Conveniently, a linking group comprising a protected amine, thiol, orcarboxyl at one end, and a phosphoramidite can be covalently attached toa hydroxyl group of a CIC. Agrawal et al. (1986) Nucleic Acids Res.14:6227-6245; Connolly (1985) Nucleic Acids Res. 13:4485-4502; Kremskyet al. (1987) Nucleic Acids Res. 15:2891-2909; Connolly (1987) NucleicAcids Res. 15:3131-3139; Bischoff et al. (1987) Anal. Biochem.164:336-344; Blanks et al. (1988) Nucleic Acids Res. 16:10283-10299; andU.S. Pat. Nos. 4,849,513, 5,015,733, 5,118,800, and 5,118,802.Subsequent to deprotection, the amine, thiol, and carboxylfunctionalities can be used to covalently attach the CIC to a peptide.Benoit et al. (1987); and Sinah et al. (1991).

A CIC-antigen conjugate can also be formed through non-covalentinteractions, such as ionic bonds, hydrophobic interactions, hydrogenbonds and/or van der Waals attractions.

Non-covalently linked conjugates can include a non-covalent interactionsuch as a biotin-streptavidin complex. A biotinyl group can be attached,for example, to a modified base of a CIC. Roget et al. (1989) NucleicAcids Res. 17:7643-7651. Incorporation of a streptavidin moiety into thepeptide portion allows formation of a non-covalently bound complex ofthe streptavidin conjugated peptide and the biotinylatedoligonucleotide.

Non-covalent associations can also occur through ionic interactionsinvolving a CIC and residues within the antigen, such as charged aminoacids, or through the use of a linker portion comprising chargedresidues that can interact with both the oligonucleotide and theantigen. For example, non-covalent conjugation can occur between agenerally negatively-charged CIC and positively-charged amino acidresidues of a peptide, e.g., polylysine, polyarginine and polyhistidineresidues.

Non-covalent conjugation between CIC and antigens can occur through DNAbinding motifs of molecules that interact with DNA as their naturalligands. For example, such DNA binding motifs can be found intranscription factors and anti-DNA antibodies.

The linkage of the CIC to a lipid can be formed using standard methods.These methods include, but are not limited to, the synthesis ofoligonucleotide-phospholipid conjugates (Yanagawa et al. (1988) NucleicAcids Symp. Ser. 19:189-192), oligonucleotide-fatty acid conjugates(Grabarek et al. (1990) Anal. Biochem. 185:131-135; and Staros et al.(1986) Anal. Biochem. 156:220-222), and oligonucleotide-sterolconjugates. Boujrad et al. (1993) Proc. Natl. Acad. Sci. USA90:5728-5731.

The linkage of the oligonucleotide to an oligosaccharide can be formedusing standard known methods. These methods include, but are not limitedto, the synthesis of oligonucleotide-oligosaccharide conjugates, whereinthe oligosaccharide is a moiety of an immunoglobulin. O'Shannessy et al.(1985) J. Applied Biochem. 7:347-355.

Additional methods for the attachment of peptides and other molecules tooligonucleotides can be found in U.S. Pat. No. 5,391,723; Kessler (1992)“Nonradioactive labeling methods for nucleic acids” in Kricka (ed.)Nonisotopic DNA Probe Techniques, Academic Press; and Geoghegan et al.(1992) Bioconjug. Chem. 3:138-146.

A CIC may be proximately associated with an antigen(s) in other ways. Insome embodiments, a CIC and antigen are proximately associated byencapsulation. In other embodiments, a CIC and antigen are proximatelyassociated by linkage to a platform molecule. A “platform molecule”(also termed “platform”) is a molecule containing sites which allow forattachment of the a CIC and antigen(s). In other embodiments, a CIC andantigen are proximately associated by adsorption onto a surface,preferably a carrier particle.

In some embodiments, the methods of the invention employ anencapsulating agent that can maintain the proximate association of the aCIC and first antigen until the complex is available to the target (orcompositions comprising such encapsulating agents). Preferably, thecomposition comprising a CIC, antigen and encapsulating agent is in theform of adjuvant oil-in-water emulsions, microparticles and/orliposomes. More preferably, adjuvant oil-in-water emulsions,microparticles and/or liposomes encapsulating a CIC are in the form ofparticles from about 0.04 μm to about 100 μm in size, preferably any ofthe following ranges: from about 0.1 μm to about 20 μm; from about 0.15μm to about 10 μm; from about 0.05 μm to about 1.00 μm; from about 0.05μm to about 0.5 μm.

Colloidal dispersion systems, such as microspheres, beads,macromolecular complexes, nanocapsules and lipid-based systems, such asoil-in-water emulsions, micelles, mixed micelles and liposomes canprovide effective encapsulation of CIC-containing compositions.

The encapsulation composition further comprises any of a wide variety ofcomponents. These include, but are not limited to, alum, lipids,phospholipids, lipid membrane structures (LMS), polyethylene glycol(PEG) and other polymers, such as polypeptides, glycopeptides, andpolysaccharides.

Polypeptides suitable for encapsulation components include any known inthe art and include, but are not limited to, fatty acid bindingproteins. Modified polypeptides contain any of a variety ofmodifications, including, but not limited to glycosylation,phosphorylation, myristylation, sulfation and hydroxylation. As usedherein, a suitable polypeptide is one that will protect a CIC-containingcomposition to preserve the immunomodulatory activity thereof. Examplesof binding proteins include, but are not limited to, albumins such asbovine serum albumin (BSA) and pea albumin.

Other suitable polymers can be any known in the art of pharmaceuticalsand include, but are not limited to, naturally-occurring polymers suchas dextrans, hydroxyethyl starch, and polysaccharides, and syntheticpolymers. Examples of naturally occurring polymers include proteins,glycopeptides, polysaccharides, dextran and lipids. The additionalpolymer can be a synthetic polymer. Examples of synthetic polymers whichare suitable for use in the present invention include, but are notlimited to, polyalkyl glycols (PAG) such as PEG, polyoxyethylatedpolyols (POP), such as polyoxyethylated glycerol (POG), polytrimethyleneglycol (PTG) polypropylene glycol (PPG), polyhydroxyethyl methacrylate,polyvinyl alcohol (PVA), polyacrylic acid, polyethyloxazoline,polyacrylamide, polyvinylpyrrolidone (PVP), polyamino acids,polyurethane and polyphosphazene. The synthetic polymers can also belinear or branched, substituted or unsubstituted, homopolymeric,co-polymers, or block co-polymers of two or more different syntheticmonomers.

The PEGs for use in encapsulation compositions of the present inventionare either purchased from chemical suppliers or synthesized usingtechniques known to those of skill in the art.

The term “LMS”, as used herein, means lamellar lipid particles whereinpolar head groups of a polar lipid are arranged to face an aqueous phaseof an interface to form membrane structures. Examples of the LMSsinclude liposomes, micelles, cochleates (i.e., generally cylindricalliposomes), microemulsions, unilamellar vesicles, multilamellarvesicles, and the like.

One colloidal dispersion system useful in the administration of CICs isa liposome. In mice immunized with a liposome-encapsulated antigen,liposomes appeared to enhance a Th1-type immune response to the antigen.Aramaki et al. (1995) Vaccine 13:1809-1814. As used herein, a “liposome”or “lipid vesicle” is a small vesicle bounded by at least one andpossibly more than one bilayer lipid membrane. Liposomes are madeartificially from phospholipids, glycolipids, lipids, steroids such ascholesterol, related molecules, or a combination thereof by anytechnique known in the art, including but not limited to sonication,extrusion, or removal of detergent from lipid-detergent complexes. Onetype of liposome for use in delivering CICs to cells is a cationicliposome. A liposome can also optionally comprise additional components,such as a tissue targeting component. It is understood that a “lipidmembrane” or “lipid bilayer” need not consist exclusively of lipids, butcan additionally contain any suitable other components, including, butnot limited to, cholesterol and other steroids, lipid-soluble chemicals,proteins of any length, and other amphipathic molecules, providing thegeneral structure of the membrane is a sheet of two hydrophilic surfacessandwiching a hydrophobic core. For a general discussion of membranestructure, see The Encyclopedia of Molecular Biology by J. Kendrew(1994). For suitable lipids see e.g., Lasic (1993) “Liposomes: fromPhysics to Applications” Elsevier, Amsterdam.

Processes for preparing liposomes containing CIC-containing compositionsare known in the art. The lipid vesicles can be prepared by any suitabletechnique known in the art. Methods include, but are not limited to,microencapsulation, microfluidization, LLC method, ethanol injection,freon injection, the “bubble” method, detergent dialysis, hydration,sonication, and reverse-phase evaporation. Reviewed in Watwe et al.(1995) Curr. Sci. 68:715-724. Techniques may be combined in order toprovide vesicles with the most desirable attributes.

The invention encompasses use of LMSs containing tissue or cellulartargeting components. Such targeting components are components of a LMSthat enhance its accumulation at certain tissue or cellular sites inpreference to other tissue or cellular sites when administered to anintact animal, organ, or cell culture. A targeting component isgenerally accessible from outside the liposome, and is thereforepreferably either bound to the outer surface or inserted into the outerlipid bilayer. A targeting component can be inter alfa a peptide, aregion of a larger peptide, an antibody specific for a cell surfacemolecule or marker, or antigen binding fragment thereof, a nucleic acid,a carbohydrate, a region of a complex carbohydrate, a special lipid, ora small molecule such as a drug, hormone, or hapten, attached to any ofthe aforementioned molecules. Antibodies with specificity toward celltype-specific cell surface markers are known in the art and are readilyprepared by methods known in the art.

The LMSs can be targeted to any cell type toward which a therapeutictreatment is to be directed, e.g., a cell type which can modulate and/orparticipate in an immune response. Such target cells and organs include,but are not limited to, APCs, such as macrophages, dendritic cells andlymphocytes, lymphatic structures, such as lymph nodes and the spleen,and nonlymphatic structures, particularly those in which dendritic cellsare found.

The LMS compositions of the present invention can additionally comprisesurfactants. Surfactants can be cationic, anionic, amphiphilic, ornonionic. A preferred class of surfactants are nonionic surfactants;particularly preferred are those that are water soluble.

In some embodiments a CIC and antigen are proximately associated bylinkage to a platform molecule, such as a proteinaceous ornon-proteinaceous (e.g., synthetic) valency platform. Examples ofsuitable platforms are described supra, in the discussion of valencyplatforms used as a spacer moiety in a CIC. Attachment of antigens tovalency platforms can be carried out using routine methods. As anexample, polypeptides contain amino acid side chain moieties withfunctional groups such as amino, carboxyl or sulfhydryl groups thatserve as sites for coupling the polypeptide to the platform. Residuesthat have such functional groups may be added to the polypeptide if thepolypeptide does not already contain these groups. Such residues may beincorporated by solid phase synthesis techniques or recombinanttechniques, both of which are well known in the peptide synthesis arts.When the polypeptide has a carbohydrate side chain(s) (or if the antigenis a carbohydrate), functional amino, sulfhydryl and/or aldehyde groupsmay be incorporated therein by conventional chemistry. For instance,primary amino groups may be incorporated by reaction of the oxidizedsugar with ethylenediamine in the presence of sodium cyanoborohydride,sulfhydryls may be introduced by reaction of cysteamine dihydrochloridefollowed by reduction with a standard disulfide reducing agent, whilealdehyde groups may be generated following periodate oxidation. In asimilar fashion, the platform molecule may also be derivatized tocontain functional groups if it does not already possess appropriatefunctional groups.

In another embodiment, a CIC and antigen are coadministered by adsorbingboth to a surface, such as a nanoparticle or microcarrier. Adsorption ofa CIC and/or antigen to a surface may occur through non-covalentinteractions, including ionic and/or hydrophobic interactions.Adsorption of polynucleotides and polypeptides to a surface for thepurpose of delivery of the adsorbed molecules to cells is well known inthe art. See, for example, Douglas et al. (1987) Crit. Rev. Ther. Drug.Carrier Syst. 3:233-261; Hagiwara et al. (1987) In Vivo 1:241-252;Bousquet et al. (1999) Pharm. Res. 16:141-147; and Kossovsky et al.,U.S. Pat. No. 5,460,831. Preferably, the material comprising theadsorbent surface is biodegradable.

In general, characteristics of nanoparticles, such as surface charge,particle size and molecular weight, depend upon polymerizationconditions, monomer concentration and the presence of stabilizers duringthe polymerization process (Douglas et al., 1987, supra). The surface ofcarrier particles may be modified, for example, with a surface coating,to allow or enhance adsorption of the CIC and/or antigen. Carrierparticles with adsorbed CIC and/or antigen may be further coated withother substances. The addition of such other substances may, forexample, prolong the half-life of the particles once administered to thesubject and/or may target the particles to a specific cell type ortissue, as described herein.

Nanocrystalline surfaces to which a CIC and antigen may be adsorbed havebeen described (see, for example, U.S. Pat. No. 5,460,831).Nanocrystalline core particles (with diameters of 1 μm or less) arecoated with a surface energy modifying layer that promotes adsorption ofpolypeptides, polynucleotides and/or other pharmaceutical agents. Asdescribed in U.S. Pat. No. 5,460,831, for example, a core particle iscoated with a surface that promotes adsorption of an oligonucleotide andis subsequently coated with an antigen preparation, for example, in theform of a lipid-antigen mixture. Such nanoparticles are self-assemblingcomplexes of nanometer sized particles, typically on the order of 0.1μm, that carry an inner layer of CIC and an outer layer of antigen.

Another adsorbent surface are nanoparticles made by the polymerizationof alkylcyanoacrylates. Alkylcyanoacrylates can be polymerized inacidified aqueous media by a process of anionic polymerization.Depending on the polymerization conditions, the small particles tend tohave sizes in the range of 20 to 3000 nm, and it is possible to makenanoparticles specific surface characteristics and with specific surfacecharges (Douglas et al., 1987, supra). For example, oligonucleotides maybe adsorbed to polyisobutyl- and polyisohexlcyanoacrylate nanoparticlesin the presence of hydrophobic cations such as tetraphenylphosphoniumchloride or quaternary ammonium salts, such as acetyltrimethyl ammoniumbromide. Oligonucleotide adsorption on these nanoparticles appears to bemediated by the formation of ion pairs between negatively chargedphosphate groups of the nucleic acid chain and the hydrophobic cations.See, for example, Lambert et al. (1998) Biochimie 80:969-976, Chavany etal. (1994) Pharm. Res. 11:1370-1378; Chavany et al. (1992) Pharm. Res.9:441-449. Polypeptides may also be adsorbed to polyalkylcyanoacrylatenanoparticles. See, for example, Douglas et al., 1987; Schroeder et al.(1998) Peptides 19:777-780.

Another adsorbent surface are nanoparticles made by the polymerizationof methylidene malonate. For example, as described in Bousquet et al.,1999, polypeptides adsorbed to poly(methylidene malonate 2.1.2)nanoparticles appear to do so initially through electrostatic forcesfollowed by stabilization through hydrophobic forces.

C. Additional Adjuvants

A CIC may also be administered in conjunction with an adjuvant.Administration of an antigen with a CIC and an adjuvant leads to apotentiation of a immune response to the antigen and thus, can result inan enhanced immune response compared to that which results from acomposition comprising the CIC and antigen alone. Adjuvants are known inthe art and include, but are not limited to, oil-in-water emulsions,water-in oil emulsions, alum (aluminum salts), liposomes andmicroparticles, including but not limited to, polystyrene, starch,polyphosphazene and polylactide/polyglycosides. Other suitable adjuvantsalso include, but are not limited to, MF59, DETOX™ (Ribi), squalenemixtures (SAF-1), muramyl peptide, saponin derivatives, mycobacteriumcell wall preparations, monophosphoryl lipid A, mycolic acidderivatives, nonionic block copolymer surfactants, Quil A, cholera toxinB subunit, polyphosphazene and derivatives, and immunostimulatingcomplexes (ISCOMs) such as those described by Takahashi et al. (1990)Nature 344:873-875, as well as, lipid-based adjuvants and othersdescribed herein. For veterinary use and for production of antibodies inanimals, mitogenic components of Freund's adjuvant (both complete andincomplete) can be used.

IV. Methods of the Invention

The invention provides methods of modulating an immune response of ananimal or population of cells, e.g., mammalian, optionally human, bloodcells (e.g., PBMCs, lymphocytes, dendritic cells), bronchial alveolarlavage cells, or other cells or cell populations containing cellsresponsive to immunostimulatory agents, by contacting the cells with aCIC or CIC-containing composition described herein (e.g., a compositioncontaining a CIC, CIC and an antigen, a CIC-antigen conjugate, aCIC/microcarrier complex, etc.) The modulation can be accomplished byany form of contacting, including without limitation, co-incubation ofcells and CIC in vitro, application of the CIC to skin of a mammal(e.g., of an experimental animal), and parenteral administration.

An immune response in animals or cell populations can be detected in anynumber of ways, including a increased expression of one or more ofIFN-γ, IFN-α, IL-2, IL-12, TNF-α, IL-6, IL-4, IL-5, IP-10, ISG-54K,MCP-1, or a change in gene expression profile characteristic of immunestimulation (see, e.g., Example 43) as well as responses such as B cellproliferation and dendritic cell maturation, The ability to stimulate animmune response in a cell population has a number of uses, e.g., in anassay system for immunosuppressive agents.

Thus, the invention provides methods of modulating an immune response inan individual, preferably a mammal, more preferably a human, comprisingadministering to the individual a CIC as described herein.Immunomodulation may include stimulating a Th1-type immune responseand/or inhibiting or reducing a Th2-type immune response. The CIC isadministered in an amount sufficient to modulate an immune response. Asdescribed herein, modulation of an immune response may be humoral and/orcellular, and is measured using standard techniques in the art and asdescribed herein.

In certain embodiments, the individual suffers from a disorderassociated with a Th2-type immune response, such as (without limitation)allergies, allergy-induced asthma, atopic dermatitis, eosinophilicgastrointestinal inflammation, eosinophilic esophagitis, and allergicbronchopulmonary aspergillosis. Administration of a CIC results inimmunomodulation, increasing levels of one or more Th1-type responseassociated cytokines, which may result in a reduction of the Th2-typeresponse features associated with the individual's response to theallergen. Immunomodulation of individuals with Th2-type responseassociated disorders results in a reduction or improvement in one ormore of the symptoms of the disorder. Where the disorder is allergy orallergy-induced asthma, improvement in one or more of the symptomsincludes a reduction one or more of the following: rhinitis, allergicconjunctivitis, circulating levels of IgE, circulating levels ofhistamine and/or requirement for ‘rescue’ inhaler therapy (e.g., inhaledalbuterol administered by metered dose inhaler or nebulizer).

In further embodiments, the individual subject to the immunomodulatorytherapy of the invention is an individual receiving a vaccine. Thevaccine may be a prophylactic vaccine or a therapeutic vaccine. Aprophylactic vaccine comprises one or more epitopes associated with adisorder for which the individual may be at risk (e.g., M. tuberculosisantigens as a vaccine for prevention of tuberculosis). Therapeuticvaccines comprise one or more epitopes associated with a particulardisorder affecting the individual, such as M. tuberculosis or M. bovissurface antigens in tuberculosis patients, antigens to which theindividual is allergic (i.e., allergy desensitization therapy) inindividuals subject to allergies, tumor cells from an individual withcancer (e.g., as described in U.S. Pat. No. 5,484,596), or tumorassociated antigens in cancer patients. The CIC may be given inconjunction with the vaccine (e.g., in the same injection or acontemporaneous, but separate, injection) or the CIC may be administeredseparately (e.g., at least 12 hours before or after administration ofthe vaccine). In certain embodiments, the antigen(s) of the vaccine ispart of the CIC, by either covalent or non-covalent linkage to the CIC.Administration of CIC therapy to an individual receiving a vaccineresults in an immune response to the vaccine that is shifted towards aTh1-type response as compared to individuals which receive vaccine notcontaining a CIC. Shifting towards a Th1-type response may be recognizedby a delayed-type hypersensitivity (DTH) response to the antigen(s) inthe vaccine, increased IFN-γ and other Th1-type response associatedcytokines, production of CTLs specific for the antigen(s) of thevaccine, low or reduced levels of IgE specific for the antigen(s) of thevaccine, a reduction in Th2-associated antibodies specific for theantigen(s) of the vaccine, and/or an increase in Th1-associatedantibodies specific for the antigen(s) of the vaccine. In the case oftherapeutic vaccines, administration of CIC and vaccine also results inamelioration of one or more symptoms of the disorder which the vaccineis intended to treat. As will be apparent to one of skill in the art,the exact symptoms and manner of their improvement will depend on thedisorder sought to be treated. For example, where the therapeuticvaccine is for tuberculosis, CIC treatment with vaccine results inreduced coughing, pleural or chest wall pain, fever, and/or othersymptoms known in the art. Where the vaccine is an allergen used inallergy desensitization therapy, the treatment results in a reduction inthe symptoms of allergy (e.g., reduction in rhinitis, allergicconjunctivitis, circulating levels of IgE, and/or circulating levels ofhistamine).

The compositions of the invention may also be used prophylactically toincrease resistance to infection by a wide range of bacterial or viralpathogens, including natural of genetically modified organisms employedas agents of biological warfare or terrorism.

Other embodiments of the invention relate to immunomodulatory therapy ofindividuals having a pre-existing disease or disorder, such as cancer oran infectious disease. Cancer is an attractive target forimmunomodulation because most cancers express tumor-associated and/ortumor specific antigens which are not found on other cells in the body.Stimulation of a Th1-type response against tumor cells results in directand/or bystander killing of tumor cells by the immune system, leading toa reduction in cancer cells and a reduction in symptoms. Administrationof a CIC to an individual having cancer results in stimulation of aTh1-type immune response against the tumor cells. Such an immuneresponse can kill tumor cells, either by direct action of cellularimmune system cells (e.g., CTLs) or components of the humoral immunesystem, or by bystander effects on cells proximal to cells targeted bythe immune system including macrophages and natural killer (NK) cells.See, for example, Cho et al. (2000) Nat. Biotechnol. 18:509-514. Intreatment of a pre-existing disease or disorder, the CIC can beadministered in conjunction with other immunotherapeutic agents such ascytokines, adjuvants and antibodies. For example, a CIC can beadministered as part of a therapeutic regimen that includesadministration of a binding agent that binds an antigen displayed bytumor cells. Exemplary binding agents include polyclonal and monoclonalantibodies. Examples of target antigens include CD20, CD22, HER2 andothers known in the art or to be discovered in the future. Withoutintending to be bound by theory, it is believed that the CIC enhanceskilling of tumor cells to which the binding agent is associated (e.g.,by enhancing antibody dependent cellular cytotoxicity and/or effectorfunction). The binding agent can optionally be labeled, e.g., with aradioisotope or toxin that damages a cell to which the binding agent isbound. The CIC may be given in conjunction with the agent (e.g., at thesame time) or before or after (e.g., less than 24 hours before or afteradministration of the agent). For example, in the case of cancer, theCIC can be administered in conjunction with a chemotherapeutic agentknown or suspected of being effective for the treatment of cancer. Asanother example, the CIC can be administered in conjunction withradiation therapy, gene therapy, or the like. The CIC may be any ofthose described herein.

Immunomodulatory therapy in accordance with the invention is alsobeneficial for individuals with infectious diseases, particularlyinfectious diseases which are resistant to humoral immune responses(e.g., diseases caused by mycobacterial infections and intracellularpathogens). Immunomodulatory therapy may be used for the treatment ofinfectious diseases caused by cellular pathogens (e.g., bacteria orprotozoans) or by subcellular pathogens (e.g., viruses). CIC therapy maybe administered to individuals suffering from mycobacterial diseasessuch as tuberculosis (e.g., M. tuberculosis and/or M. bovis infections),leprosy (i.e., M. leprae infections), or M. marinum or M. ulceransinfections. CIC therapy is also may also be used for the treatment ofviral infections, including infections by influenza virus, respiratorysyncytial virus (RSV), hepatitis virus B, hepatitis virus C, herpesviruses, particularly herpes simplex viruses, and papilloma viruses.Diseases caused by intracellular parasites such as malaria (e.g.,infection by Plasmodium vivax, P. ovale, P. falciparum and/or P.malariae), leishmaniasis (e.g., infection by Leishmania donovani, L.tropica, L. mexicana, L. braziliensis, L. peruviana, L. infantum, L.chagasi, and/or L. aethiopica), and toxoplasmosis (i.e., infection byToxoplasmosis gondii) also benefit from CIC therapy. CIC therapy mayalso be used for treatment of parasitic diseases such as schistosomiasis(i.e., infection by blood flukes of the genus Schistosoma such as S.haematobium, S. mansoni, S. japonicum, and S. mekongi) and clonorchiasis(i.e., infection by Clonorchis sinensis). Administration of a CIC to anindividual suffering from an infectious disease results in anamelioration of symptoms of the infectious disease. In some embodiments,the infectious disease is not a viral disease.

The invention further provides methods of increasing or stimulating atleast one Th1-associated cytokine in an individual, including IL-2,IL-12, TNF-α, TNF-β, IFN-γ and IFN-α. In certain embodiments, theinvention provides methods of increasing or stimulating IFN-γ in anindividual, particularly in an individual in need of increased IFN-γlevels, by administering an effective amount of a CIC to the individual.Individuals in need of increased IFN-γ are those having disorders whichrespond to the administration of IFN-γ. Such disorders include a numberof inflammatory disorders including, but not limited to, ulcerativecolitis. Such disorders also include a number of fibrotic disorders,including, but not limited to, idiopathic pulmonary fibrosis (IPF),scleroderma, cutaneous radiation-induced fibrosis, hepatic fibrosisincluding schistosomiasis-induced hepatic fibrosis, renal fibrosis aswell as other conditions which may be improved by administration ofIFN-γ. An increase in IFN-γ levels may result in amelioration of one ormore symptoms, stabilization of one or more symptoms, or prevention ofprogression (e.g., reduction or elimination of additional lesions orsymptoms) of the disorder which responds to IFN-γ. The methods of theinvention may be practiced in combination with other therapies whichmake up the standard of care for the disorder, such as administration ofanti-inflammatory agents such as systemic corticosteroid therapy (e.g.,cortisone) in IPF.

In certain embodiments, the invention provides methods of increasingIFN-α in an individual, particularly in an individual in need ofincreased IFN-α levels, by administering an effective amount of a CIC tothe individual such that IFN-α levels are increased. Individuals in needof increased IFN-α are those having disorders which respond to theadministration of IFN-α, including recombinant IFN-α, including, but notlimited to, viral infections and cancer.

Administration of a CIC in accordance with certain embodiments of theinvention results in an increase in IFN-α levels, and results inamelioration of one or more symptoms, stabilization of one or moresymptoms, or prevention of progression (e.g., reduction or eliminationof additional lesions or symptoms) of the disorder which responds toIFN-α. The methods of the invention may be practiced in combination withother therapies which make up the standard of care for the disorder,such as administration of anti-viral agents for viral infections.

As will be apparent upon review of this disclosure, the spacercomposition of a CIC can affect the immune response elicited byadministration of the CIC. Virtually all of the spacers tested (with theexception of dodecyl) can be used in CICs to efficiently induce IFN-γ inhuman PBMCs. However, the spacer composition of linear CICs has beenobserved to have differential effects on induction of IFN-α. Forexample, CICs containing, for example, HEG, TEG or C6 spacers tend tocause higher IFN-α induction (and reduced B cell proliferation) in PBMCsthan did CICs containing C3, C4 or abasic spacers (see, e.g.: Example34, infra).

The invention also provides methods of reducing levels, particularlyserum levels, of IgE in an individual having an IgE-related disorder byadministering an effective amount of a CIC to the individual. In suchmethods, the CIC may be administered alone (e.g., without antigen) oradministered with antigen, such as an allergen. An IgE-related disorderis a condition, disorder, or set of symptoms ameliorated by a reductionin IgE levels. Reduction in IgE results in an amelioration of symptomsof the IgE-related disorder. Such symptoms include allergy symptoms suchas rhinitis, conjunctivitis, in decreased sensitivity to allergens, areduction in the symptoms of allergy in an individual with allergies, ora reduction in severity of an allergic response.

Guided by the present disclosure, CICs can be designed to achievespecific desired physiological responses. For example, the IFN-αinducing activity, and B cell proliferation-inducing activities of CICscan be independently varied based on the structure of the CIC andselection of NAMs. For example, as illustrated in FIG. 10, IFN-αproduction was effectively stimulated by CICs containing the sequences5′-TCGXCGX and 5′-TCGXTCG (e.g., ^(F)5′-TCGXCGX and ^(F)5′-TCGXTCG)where X is any nucleotide. (It will be appreciated by the reader thatother CIC structures can also effectively stimulate IFN-α production.)In addition, induction of IFN-α was significantly enhanced bymultivalent CICs that present multiple copies NAMs of the aforementionedmotifs linked to long, hydrophilic spacer moieties (e.g., hexaethyleneglycol).

The effect of CIC structure (including nucleic acid moiety motifs andspacers) on B cell proliferation was also tested using purifiedperipheral blood B cells. Dose titration was performed in order todetermine the optimal concentration for the assay, which was determinedto be 5 mg/ml (data not shown). CICs containing heptameric motifs with a5′-TCGT induced the highest levels of proliferation, while,surprisingly, many CICs containing 5′-TCGA sequences stimulated only lowlevels or background levels of proliferation. Comparing CICs containingidentical branched CIC structures, spacers, and sequences, with theexception of the nucleotide following the 5′-TCG in each motif,confirmed that TCGT sequences were significantly more active than TCGAsequences, with TCGC and TCGG sequences having intermediate activity.The bases following the 5′-TCGX also had some influence on B cellactivity. We found that C-74, containing the nucleic acid moiety motifTCGATTT, induced moderate levels of B cell proliferation, which wereintermediate between the low to background levels found for otherTCGA-containing CICs and the levels observed for C-41, containing thesequence TCGTTTT, and other TCGT-containing CICs. No significantdifferences in proliferation were observed for linear (C-21) vs.branched CICs (C-94) or for CICs containing different types of spacers(compare C-94, C-103, and C-104). From these data it appears thatstimulation of B cell activity is largely a function of the sequence ofthe nucleic acid moieties, with spacers and multimeric presentationbeing less significant.

The data presented in FIGS. 10 and 11 illustrate of the ability toindependently vary B cell proliferation-inducing and IFN-α-inducingactivity. For example, C-101, C-125, and C-145 all induce high levels ofIFN-α, but induce very little B cell proliferation. C-94, C-142, andC-158 induce high levels of IFN-α, and also induce B cells toproliferate. Finally, C-104 induces no measurable IFN-α, but stimulatesB cells to proliferate.

Because the IFN-α inducing activity and B cell proliferation-inducingactivities of CICs can be independently varied based on the structure ofthe CIC and selection of NAMs it is possible to identify and produceCICs with different levels of each of these activities, using screeningmethods described herein and the information about B cell stimulatingactivity described herein. For example, CICs can be designed to exhibitdifferent B cell proliferation-inducing activities, from insignificantup to levels equivalent to P-6, independently of the amount of IFN-αinduced by that CIC. Similarly, CICs with different levels ofIFN-inducing activity can be identified and produced.

Thus, without limitation, in one aspect, the invention provides CICsthat induce IFN-α production and do not induce human B cellproliferation, and methods of using such CICs. In a related aspect, theinvention provides CICs that induce IFN-α production and little human Bcell proliferation and methods of using such CICs. In a related aspect,the invention provides design algorithms and screening methods foridentifying CICs with these properties.

A CIC is considered to not induce human B cell proliferation if B cellproliferation in the presence of the CIC is at “background” levels,i.e., 0 to 15%, optionally 0 to about 10%, of the proliferation inducedby an equal amount (e.g., 5 μg/ml) of P-6. A CIC is considered to induce“little” human B cell proliferation if B cell proliferation in thepresence of the CIC is between greater than 15 to about 30% of P-6.Thus, in some embodiments a CIC of the invention induces less than about30%, sometimes less then about 25%, sometimes less than about 20%,sometimes less than about 15% or less than about 10% of the level of Bcell proliferation induced by P-6. For illustration and not limitation,examples of such CICs include: C-51; C-101; C-144; C-145; C-146; C-148;C-149; C-150. Alternatively, a CIC is considered to not induce human Bcell proliferation if B cell proliferation in the presence of the CIC isnot statistically significantly greater that the B cell proliferationinduced by an equal concentration (e.g., 5 ug/ml) of a control chimericcompound, such as M-3, using an in vitro assay. See FIG. 11.

The ability to “program” CICs to exhibit different biological propertiesallows for the assembly of CICs exhibiting a defined set of activitiestailored for specific clinical applications. For example, CICs with highIFN-α production and little B cell activation may be particularly usefulin cancer therapies, while CICs with moderate IFN-α production andlittle B cell activation are particularly useful for treatment ofdiseases such as asthma. As previously noted, for certain indications,including the treatment of allergic asthma and certain cancers, it maybe desirable to avoid polyclonal B cell activation, which might resultin the potentiation of asthma-mediating B cells or B cell lymphomas. Avariety of uses are known for CICs that preferentially stimulate B cellproliferation, including without limitation in vivo expansion to produceB cell clones for analysis.

Methods of the invention includes embodiments in which CICs areadministered in the form of a CIC/microcarrier complex(s).

In some embodiments, the invention provides methods of stimulating CTLproduction in an individual, comprising administering an effectiveamount of a CIC to the individual such that CTL production is increased.

As will be apparent to one of skill in the art, the methods of theinvention may be practiced in combination with other therapies for theparticular indication for which the CIC is administered. For example,CIC therapy may be administered in conjunction with anti-malarial drugssuch as chloroquine for malaria patients, in conjunction withleishmanicidal drugs such as pentamidine and/or allopurinol forleishmaniasis patients, in conjunction with anti-mycobacterial drugssuch as isoniazid, rifampin and/or ethambutol in tuberculosis patients,or in conjunction with allergen desensitization therapy for atopic(allergy) patients.

A. Administration and Assessment of the Immune Response

The CIC can be administered in combination with pharmaceutical and/orimmunogenic and/or other immunostimulatory agents, as described herein,and can be combined with a physiologically acceptable carrier thereof.

For example, a CIC or composition of the invention can be administeredin conjunction with other immunotherapeutic agents such as cytokines,adjuvants and antibodies. The CIC may be given in conjunction with theagent (e.g., at the same time, or before or after (e.g., less than 24hours before or after administration of the agent). The CIC may be anyof those described herein.

As with all immunostimulatory compositions, the immunologicallyeffective amounts and method of administration of the particular CICformulation can vary based on the individual, what condition is to betreated and other factors evident to one skilled in the art. Factors tobe considered include the presence of a coadministered antigen, whetheror not the CIC will be administered with or covalently attached to anadjuvant or delivery molecule, route of administration and the number ofimmunizing doses to be administered. Such factors are known in the artand it is well within the skill of those in the art to make suchdeterminations without undue experimentation. A suitable dosage range isone that provides the desired modulation of immune response to theantigen. Generally, dosage is determined by the amount of CICadministered to the patient, rather than the overall quantity of CIC.Exemplary dosage ranges of the CIC, given in amounts of CIC delivered,may be, for example, from about any of the following: 1 to 500 μg/kg,100 to 400 μg/kg, 200 to 300 μg/kg, 1 to 100 μg/kg, 100 to 200 μg/kg,300 to 400 μg/kg, 400 to 500 μg/kg. The absolute amount given to eachpatient depends on pharmacological properties such as bioavailability,clearance rate and route of administration.

The effective amount and method of administration of the particular CICformulation can vary based on the individual patient and the stage ofthe disease and other factors evident to one skilled in the art. Theroute(s) of administration suited for a particular application will beknown to one of skill in the art. Routes of administration include butare not limited to topical, dermal, transdermal, transmucosal,epidermal, parenteral, gastrointestinal, and naso-pharyngeal andpulmonary, including transbronchial and transalveolar. A suitable dosagerange is one that provides sufficient CIC-containing composition toattain a tissue concentration of about 1-10 μM as measured by bloodlevels. The absolute amount given to each patient depends onpharmacological properties such as bioavailability, clearance rate androute of administration.

As described herein, APCs and tissues with high concentration of APCsare preferred targets for the CIC. Thus, administration of CIC tomammalian skin and/or mucosa, where APCs are present in relatively highconcentration, is preferred.

The present invention provides CIC formulations suitable for topicalapplication including, but not limited to, physiologically acceptableimplants, ointments, creams, rinses and gels. Topical administration is,for instance, by a dressing or bandage having dispersed therein adelivery system, by direct administration of a delivery system intoincisions or open wounds, or by transdermal administration devicedirected at a site of interest. Creams, rinses, gels or ointments havingdispersed therein a CIC are suitable for use as topical ointments orwound filling agents.

Preferred routes of dermal administration are those which are leastinvasive. Preferred among these means are transdermal transmission,epidermal administration and subcutaneous injection. Of these means,epidermal administration is preferred for the greater concentrations ofAPCs expected to be in intradermal tissue.

Transdermal administration is accomplished by application of a cream,rinse, gel, etc. capable of allowing the CIC to penetrate the skin andenter the blood stream. Compositions suitable for transdermaladministration include, but are not limited to, pharmaceuticallyacceptable suspensions, oils, creams and ointments applied directly tothe skin or incorporated into a protective carrier such as a transdermaldevice (so-called “patch”). Examples of suitable creams, ointments etc.can be found, for instance, in the Physician's Desk Reference.

For transdermal transmission, iontophoresis is a suitable method.Iontophoretic transmission can be accomplished using commerciallyavailable patches which deliver their product continuously throughunbroken skin for periods of several days or more. Use of this methodallows for controlled transmission of pharmaceutical compositions inrelatively great concentrations, permits infusion of combination drugsand allows for contemporaneous use of an absorption promoter.

An exemplary patch product for use in this method is the LECTRO PATCHtrademarked product of General Medical Company of Los Angeles, Calif.This product electronically maintains reservoir electrodes at neutral pHand can be adapted to provide dosages of differing concentrations, todose continuously and/or periodically. Preparation and use of the patchshould be performed according to the manufacturer's printed instructionswhich accompany the LECTRO PATCH product; those instructions areincorporated herein by this reference. Other occlusive patch systems arealso suitable.

For transdermal transmission, low-frequency ultrasonic delivery is alsoa suitable method. Mitragotri et al. (1995) Science 269:850-853.Application of low-frequency ultrasonic frequencies (about 1 MHz) allowsthe general controlled delivery of therapeutic compositions, includingthose of high molecular weight.

Epidermal administration essentially involves mechanically or chemicallyirritating the outermost layer of the epidermis sufficiently to provokean immune response to the irritant. Specifically, the irritation shouldbe sufficient to attract APCs to the site of irritation.

An exemplary mechanical irritant means employs a multiplicity of verynarrow diameter, short tines which can be used to irritate the skin andattract APCs, to the site of irritation, to take up CIC transferred fromthe end of the tines. For example, the MONO-VACC old tuberculin testmanufactured by Pasteur Merieux of Lyon, France contains a devicesuitable for introduction of CIC-containing compositions.

The device (which is distributed in the U.S. by Connaught Laboratories,Inc. of Swiftwater, Pa.) consists of a plastic container having asyringe plunger at one end and a tine disk at the other. The tine disksupports a multiplicity of narrow diameter tines of a length which willjust scratch the outermost layer of epidermal cells. Each of the tinesin the MONO-VACC kit is coated with old tuberculin; in the presentinvention, each needle is coated with a pharmaceutical composition of aCIC formulation. Use of the device is preferably according to themanufacturer's written instructions included with the device product.Similar devices which can also be used in this embodiment are thosewhich are currently used to perform allergy tests.

Another suitable approach to epidermal administration of CIC is by useof a chemical which irritates the outermost cells of the epidermis, thusprovoking a sufficient immune response to attract APCs to the area. Anexample is a keratinolytic agent, such as the salicylic acid used in thecommercially available topical depilatory creme sold by NoxemaCorporation under the trademark NAIR. This approach can also be used toachieve epithelial administration in the mucosa. The chemical irritantcan also be applied in conjunction with the mechanical irritant (as, forexample, would occur if the MONO-VACC type tine were also coated withthe chemical irritant). The CIC can be suspended in a carrier which alsocontains the chemical irritant or coadministered therewith.

Parenteral routes of administration include but are not limited toelectrical (iontophoresis) or direct injection such as direct injectioninto a central venous line, intravenous, intramuscular, intraperitoneal,intradermal, or subcutaneous injection. Formulations of CIC suitable forparenteral administration are generally formulated in USP water or waterfor injection and may further comprise pH buffers, salts bulking agents,preservatives, and other pharmaceutically acceptable excipients. CICsfor parenteral injection may be formulated in pharmaceuticallyacceptable sterile isotonic solutions such as saline and phosphatebuffered saline for injection.

Gastrointestinal routes of administration include, but are not limitedto, ingestion and rectal. The invention includes formulations CICsuitable for gastrointestinal administration including, but not limitedto, pharmaceutically acceptable powders, pills or liquids for ingestionand suppositories for rectal administration. As will be apparent to oneof skill in the art, pills or suppositories will further comprisepharmaceutically acceptable solids, such as starch, to provide bulk forthe composition.

Naso-pharyngeal and pulmonary administration include are accomplished byinhalation, and include delivery routes such as intranasal,transbronchial and transalveolar routes. The invention includesformulations of CIC suitable for administration by inhalation including,but not limited to, liquid suspensions for forming aerosols as well aspowder forms for dry powder inhalation delivery systems. Devicessuitable for administration by inhalation of CIC formulations include,but are not limited to, atomizers, vaporizers, nebulizers, and drypowder inhalation delivery devices.

The choice of delivery routes can be used to modulate the immuneresponse elicited. For example, IgG titers and CTL activities wereidentical when an influenza virus vector was administered viaintramuscular or epidermal (gene gun) routes; however, the muscularinoculation yielded primarily IgG2a, while the epidermal route yieldedmostly IgG1. Pertmer et al. (1996) J. Virol. 70:6119-6125. Thus, oneskilled in the art can take advantage of slight differences inimmunogenicity elicited by different routes of administering theimmunomodulatory oligonucleotides of the present invention.

The above-mentioned compositions and methods of administration are meantto describe but not limit the methods of administering the formulationsof CIC of the invention. The methods of producing the variouscompositions and devices are within the ability of one skilled in theart and are not described in detail here.

Analysis (both qualitative and quantitative) of the immune response toCIC can be by any method known in the art, including, but not limitedto, measuring antigen-specific antibody production (including measuringspecific antibody+subclasses), activation of specific populations oflymphocytes such as CD4+ T cells, NK cells or CTLs, production ofcytokines such as IFN-γ, IFN-α, IL-2, IL-4, IL-5, IL-10 or IL-12 and/orrelease of histamine. Methods for measuring specific antibody responsesinclude enzyme-linked immunosorbent assay (ELISA) and are well known inthe art. Measurement of numbers of specific types of lymphocytes such asCD4+ T cells can be achieved, for example, with fluorescence-activatedcell sorting (FACS). Cytotoxicity and CTL assays can be performed forinstance as described in Raz et al. (1994) Proc. Natl. Acad. Sci. USA91:9519-9523 and Cho et al. (2000). Cytokine concentrations can bemeasured, for example, by ELISA. These and other assays to evaluate theimmune response to an immunogen are well known in the art. See, forexample, SELECTED METHODS IN CELLULAR IMMUNOLOGY (1980) Mishell andShiigi, eds., W.H. Freeman and Co.

Preferably, a Th1-type response is stimulated, i.e., elicited and/orenhanced. With reference to the invention, stimulating a Th1-type immuneresponse can be determined in vitro or ex vivo by measuring cytokineproduction from cells treated with a CIC as compared to control cellsnot treated with CIC. Methods to determine the cytokine production ofcells include those methods described herein and any known in the art.The type of cytokines produced in response to CIC treatment indicate aTh1-type or a Th2-type biased immune response by the cells. As usedherein, the term “Th1-type biased” cytokine production refers to themeasurable increased production of cytokines associated with a Th1-typeimmune response in the presence of a stimulator as compared toproduction of such cytokines in the absence of stimulation. Examples ofsuch Th1-type biased cytokines include, but are not limited to, IL-2,IL-12, IFN-γ and IFN-α. In contrast, “Th2-type biased cytokines” refersto those associated with a Th2-type immune response, and include, butare not limited to, IL-4, IL-5, and IL-13. Cells useful for thedetermination of CIC activity include cells of the immune system,primary cells isolated from a host and/or cell lines, preferably APCsand lymphocytes, even more preferably macrophages and T cells.

Stimulating a Th1-type immune response can also be measured in a hosttreated with a CIC can be determined by any method known in the artincluding, but not limited to: (1) a reduction in levels of IL-4 or IL-5measured before and after antigen-challenge; or detection of lower (oreven absent) levels of IL-4 or IL-5 in a CIC treated host as compared toan antigen-primed, or primed and challenged, control treated withoutCIC; (2) an increase in levels of IL-12, IL-18 and/or IFN (α, β or γ)before and after antigen challenge; or detection of higher levels ofIL-12, IL-18 and/or IFN (α, β or γ) in a CIC treated host as compared toan antigen-primed or, primed and challenged, control treated withoutCIC; (3) “Th1-type biased” antibody production in a CIC treated host ascompared to a control treated without CIC; and/or (4) a reduction inlevels of antigen-specific IgE as measured before and after antigenchallenge; or detection of lower (or even absent) levels ofantigen-specific IgE in a CIC treated host as compared to anantigen-primed, or primed and challenged, control treated without CIC. Avariety of these determinations can be made by measuring cytokines madeby APCs and/or lymphocytes, preferably macrophages and/or T cells, invitro or ex vivo using methods described herein or any known in the art.Some of these determinations can be made by measuring the class and/orsubclass of antigen-specific antibodies using methods described hereinor any known in the art.

The class and/or subclass of antigen-specific antibodies produced inresponse to CIC treatment indicate a Th1-type or a Th2-type biasedimmune response by the cells. As used herein, the term “Th1-type biased”antibody production refers to the measurable increased production ofantibodies associated with a Th1-type immune response (i.e.,Th1-associated antibodies). One or more Th1 associated antibodies may bemeasured. Examples of such Th1-type biased antibodies include, but arenot limited to, human IgG1 and/or IgG3 (see, e.g., Widhe et al. (1998)Scand. J. Immunol. 47:575-581 and de Martino et al. (1999) Ann. AllergyAsthma Immunol. 83:160-164) and murine IgG2a. In contrast, “Th2-typebiased antibodies” refers to those associated with a Th2-type immuneresponse, and include, but are not limited to, human IgG2, IgG4 and/orIgE (see, e.g., Widhe et al. (1998) and de Martino et al. (1999)) andmurine IgG1 and/or IgE.

The Th1-type biased cytokine induction which occurs as a result ofadministration of CIC produces enhanced cellular immune responses, suchas those performed by NK cells, cytotoxic killer cells, Th1 helper andmemory cells. These responses are particularly beneficial for use inprotective or therapeutic vaccination against viruses, fungi, protozoanparasites, bacteria, allergic diseases and asthma, as well as tumors.

In some embodiments, a Th2 response is suppressed. Suppression of a Th2response may be determined by, for example, reduction in levels ofTh2-associated cytokines, such as IL-4 and IL-5, as well as IgEreduction and reduction in histamine release in response to allergen.

V. Kits of the Invention

The invention provides kits. In certain embodiments, the kits of theinvention comprise one or more containers comprising a CIC. The kits mayfurther comprise a suitable set of instructions, generally writteninstructions, relating to the use of the CIC for the intended treatment(e.g., immunomodulation, ameliorating symptoms of an infectious disease,increasing IFN-γ levels, increasing IFN-α levels, or ameliorating anIgE-related disorder).

The kits may comprise CIC packaged in any convenient, appropriatepackaging. For example, if the CIC is a dry formulation (e.g., freezedried or a dry powder), a vial with a resilient stopper is normallyused, so that the CIC may be easily resuspended by injecting fluidthrough the resilient stopper. Ampoules with non-resilient, removableclosures (e.g., sealed glass) or resilient stoppers are mostconveniently used for liquid formulations of CIC. Also contemplated arepackages for use in combination with a specific device, such as aninhaler, nasal administration device (e.g., an atomizer) or an infusiondevice such as a minipump.

The instructions relating to the use of CIC generally includeinformation as to dosage, dosing schedule, and route of administrationfor the intended treatment. The containers of CIC may be unit doses,bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable.

In some embodiments, the kits further comprise an antigen (or one ormore antigens), which may or may not be packaged in the same container(formulation) as the CIC(s). Antigen have been described herein.

In certain embodiments, the kits of the invention comprise a CIC in theform of a CIC/microcarrier complex (CIC/MC) and may further comprise aset of instructions, generally written instructions, relating to the useof the CIC/MC complex for the intended treatment (e.g.,immunomodulation, ameliorating symptoms of an infectious disease,increasing IFN-γ levels, increasing IFN-α levels, or ameliorating anIgE-related disorder).

In some embodiments, kits of the invention comprise materials forproduction of CIC/MC complex generally include separate containers ofCIC and MC, although in certain embodiments materials for producing theMC are supplied rather than preformed MC. The CIC and MC are preferablysupplied in a form which allows formation of CIC/MC complex upon mixingof the supplied CIC and MC. This configuration is preferred when theCIC/MC complex is linked by non-covalent bonding. This configuration isalso preferred when the CIC and MC are to be crosslinked via aheterobifunctional crosslinker; either CIC or the MC is supplied in an“activated” form (e.g., linked to the heterobifunctional crosslinkersuch that a moiety reactive with the CIC is available).

Kits for CIC/MC complexes comprising a liquid phase MC preferablycomprise one or more containers including materials for producing liquidphase MC. For example, a CIC/MC kit for oil-in-water emulsion MC maycomprise one or more containers containing an oil phase and an aqueousphase. The contents of the container are emulsified to produce the MC,which may be then mixed with the CIC, preferably a CIC which has beenmodified to incorporate a hydrophobic moiety. Such materials include oiland water, for production of oil-in-water emulsions, or containers oflyophilized liposome components (e.g., a mixture of phospholipid,cholesterol and a surfactant) plus one or more containers of an aqueousphase (e.g., a pharmaceutically-acceptable aqueous buffer).

VI. EXAMPLES

The following Examples are provided to illustrate, but not limit, theinvention.

Example 1 Structure of Polynucleotides and Chimeric Compounds

Table 2 shows the structures of polynucleotides and chimeric moleculesreferred to in the Examples. “HEG” is a hexa(ethylene glycol) spacermoiety; “TEG” is triethylene glycol; “C3” is a propyl spacer moiety;“C4” is a butyl spacer; “C6” is a hexyl spacer; “C12” is a dodecylspacer; “HME” is 2-hydroxymethylethyl; “abasic” or “ab” is1′2′-dideoxyribose. Other spacers are described in this specificationand in the figures.

Except where noted in Table 2 or in specific examples, all nucleotidelinkages and linkages between nucleic acid moieties and spacer moietiesare phosphorothioate ester. For example, in CICs comprising compound(multiple subunits) spacer moieties with multiple HEG or C3 units (e.g.,C-13, C-14, C-15, C-15, C-91, C-92, C-36, C-37, and C-38) the C3 or HEGunits are linked with a phosphorothiate linker. Similarly, the branchedCICs shown (e.g., C-93, C-94, C-95, C-96, C-97, C-98, C-100, C-101,C-103, C-104, C-121, C-122, C-123, C-124, C-125, C-126, C-127, C-129,C-130) comprise phosphorothioate linkers between the branching subunitand the linear subunit of the spacer. Other branched CICs shown (e.g.,C-26, C-99, C-102, C-105, and C-137) are prepared by conjugationstrategies and have linking groups as described in the Examples.

Table 2 also includes CICs (e.g., C-128, C-106-C-113) with an endlinking group (e.g., HS(CH₂)₆— and HO(CH₂)₆SS(CH₂)₆) used to link thesemolecules with branched spacer moieties to create branched CICs. See,e.g., Example 18. These linking groups are connected to the CIC with aphosphorothioate linkage.

TABLE 2 TEST COMPOUNDS AND POLYNUCLEOTIDES Compound DesignationNumber(s) Structure P-1 5′-TCGTCGA-3′ P-2 5′-TCGTCG-3′ P-3 5′-ACGTTCG-3′P-4 5′-AGATGAT-3′ P-5 5′-ATCTCGA-3′ P-65′-TGA CTG TGA ACG TTC GAG ATG A-3′ (SEQ ID NO: 2) P-75′-TGA CTG TGA ACC TTA GAG ATG A-3′ (SEQ ID NO: 3) P-85′-TGACTGTGAAGGTTAGAGATGA-3′ (SEQ ID NO: 136) P-95′-CTGTGAACGTTCGAGATG-3′ (SEQ ID NO: 83) P-10 5′-TCGTCGAACGTTCGAGATG-3′(SEQ ID NO: 41) P-11 5′-AACGTT-3′ P-12 5′-TCGTCGT-3′ P-13 5′-TCGAGAT-3′P-14 5′-TCGACGT-3′ P-15 HO(CH₂)₆SS(CH₂)₆-5′-TGACTGTGAACCTTAGAGATGA-3′(SEQ ID NO: 137) P-16 HS(CH₂)₆-5′-TGACTGTGAACCTTAGAGATGA-3′(SEQ ID NO: 138) P-17 5′-TCGAACGTTCGA-3′ (SEQ ID NO: 155) M-15′-TGCTGC-3′-HEG-5′-AGCTTGC-3′-HEG-5′-AGATGAT-3′ M-25′-TCCTCCA-3′-HEG-5′-ACCTTAG-3′-HEG-5′-AGATGAT-3′ M-3(5′-TAGTCAT-3′-HEG)₂-glycerol-HEG-5′-AACCTTC-3′ M-17(5′-TAGTCAT-3′-HEG)₂-symmetrical doubler-HEG-5′-TAGTCAT-3′ M-18(5′-TAGTCAT-3′-HEG)₃-trebler-HEG-5′-TAGTCAT-3′ M-19(5′-TAGTCAT-3′-TEG)₂-glycerol-TEG-5′-TAGTCAT-3′ M-20(5′-TAGTCAT-3′-C3)₂-glycerol-C3-5′-TAGTCAT-3′ M-215′-TCCTCCA-3′-HEG-5′-ACCTTAG-3′-HEG-5′-AGATGAT-C₆NH₂ M-22(5′-TAGTCAT-3′-HEG)₂-glycerol-HEG-5′-AACCTTC-3′-C₆NH₂ C-85′-TCGTCGA-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′ C-95′-TCGTCGA-3′-C3-5′-ACGTTCG-3′-C3-5′-AGATGAT-3′ C-105′-TCGTCG-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′ C-115′-TCGTCG-3′-C3-5′-ACGTTCG-3′-C3-5′-AGATGAT-3′ C-12(5′-TCGTCGA-3′)₂-glycerol-3′-AGCTGCT-5′ C-13 5′-TCGTCG-3′-(C3)₁₅-5′-T-3′C-14 5′-TCGTCG-3′-(HME)₁₅-5′-T-3′ C-15 5′-TCGTCG-3′-(TEG)₈-5′-T-3′ C-165′-TCGTCG-3′-(HEG)₄-5′-T-3′ C-175′-TCGTCG-3′-C4-5′-ACGTTCG-3′-C4-5′-AGATCAT-3′ C-185′-TCGTCG-3′-TEG-5′-ACGTTCG-3′-TEG-5′-AGATGAT-3′ C-195′-TCGTCG-3′-C12-5′-ACGTTCG-3′-C12-5′-AGATGAT-3′ C-205′-TCGTCG-3′-abasic-5′-ACGTTCG-3′-abasic-5′-AGATCAT-3′ C-215′-TCGTCGA-3′-HEG-5′-TCGTCGA-3′-HEG-5′-TCGTCGA-3′ C-225′-TCGTCG-3′-HEG-5′-TCGTCG-3′-HEG-5′-TCGTCG-3′ C-235′-TCGTCG-3′-HEG-5′-AACGTT-3′-HEG-5′-AGATGAT-3′ C-245′-ACGTTCG-3′-HEG-5′-ACGTTCG-3′-HEG-5′AGATCAT-3′ C-255′-TCGTCG-3′-HME-5′-ACGTTCG-3′-HME-5′-AGATCAT-3′ C-26(5′-TCGTCGA-3′)₄-R where R = Starburst Dendrimer ® (See Ex. 18) C-27(5′-TCGTCGA-3′)₂-glycerol-5′-AACGTTC-3′ C-28(5′-TCGTCGA-3′)₂-glycerol-5′-TCGTCGA-3′ C-295′-TCGTCG-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATCAT-3′-TEG C-30HEG-5′-TCGTCG-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′-TEG C-31HEG-5′-TCGTCG-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′-TEG(phosphodiester linkages) C-325′-TCG-3′-HEG-5′-TCG-3′-HEG-5′-TCG-3′-HEG-5′-TCG-3′-HEG-5′-TCG-3′-HEG-5′-TCG-3′C-33 5′-TCGTCGA-3′-C3-5′-TCGTCGA-3′-C3-5′-TCGTCGA-3′all phosphorothioate linkages C-34HC(CH₂)₆-5′-TCGTCGA-3′-C3-5′-ACGTTCG-3′-C3-5′-AGATGAT-3′ C-35

C-36 5′-TCGTCGA-3′-(HEG)₆-5′-TCGTCGA-3′ (phosphodiester linkages) C-375′-TCGTCGA-3′-(HEG)₄-3′-AGCTGCT-5′ C-385′-TCGTCGA-3′-(HEG)₄-5′-TCGTCGA-3′ C-39 5′-TCGTCGA-3′-HEG-5′-TCGTCGA-3′C-40 5′-TCGTCG-3′-HEG-5′-TCGA-3′ C-415′-TCGTTTT-3′-HEG-5′-TCGTTTT-3′-HEG-5′-TCGTTTT-3′ C-425′-TCGTCGT-3′-HEG-5′-TCGTCGT-3′-HEG-5′-TCGTCGT-3′ C-435′-TCGTC-3′-HEG-5′-TCGTC-3′-HEG-5′-TCGTC-3′-HEG-5′-TCGTC-3′ C-445′-TCGT-3′-HEG-5′-TCGT-3′-HEG-5′-TCGT-3′-HEG-5′-TCGT-3′-HEG-5′-TCGT-3′C-45 5′-TCGAGAT-3′-HEG-5′-TCGAGAT-3′-HEG-5′-TCGAGAT-3′ C-465′-TTCGTTT-3′-HEG-5′-TTCGTTT-3′-HEG-5′-TTCGTTT-3′ C-475′-TCGTCGT-3′-HEG-5′-TGTCGTT-3′-HEG-5′-TGTCGTT-3′ C-485′-TCGTCGA-3′-HEG-5′-ACGTTCG-3′-HEG-5′-TCGTCGA-3′ C-495′-TCGTCGA-3′-HEG-5′-ACGTTCG-3′-HEG-5′-GGGGGG-3′ C-505′-TCGAACG-3′-HEG-5′-TCGAACG-3′-HEG-5′-TCGAACG-3′ C-515′-TCGACGT-3′-HEG-5′-TCGACGT-3′-HEG-5′-TCGACGT-3′ C-525′-CGTTCGA-3′-HEG-5′-CGTTCGA-3′-HEG-5′-CGTTCGA-3′ C-535′-TGACTGTGA-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′ C-545′-TCGTCGA-3′-HEG-5′AACGTTC-3′-HEG-5′-AGATGAT-3′ C-555′-TCGTCGA-3′-HEG-5′-AACGTTC-3′-HEG-5′-TCGTCGA-3′ C-565′-TCGTCGA-3′-HEG-5′-AGATGAT-3′-HEG-5′-ACGTTCG-3′ C-575′-ACGTTCG-3′-HEG-5′-TCGTCGA-3′-HEG-5′-AGATGAT-3′ C-585′-ACGTTCG-3′-HEG-5′-AGATGAT-3′-HEG-5′-TCGTCGA-3′ C-595′-AGATGAT-3′-HEG-5′-TCGTCGA-3′-HEG-5′-ACGTTCG-3′ C-605′-AGATGAT-3′-HEG-5′-ACGTTCG-3′-HEG-5′-TCGTCGA-3′ C-615′-TCCATTT-3′-HEG-5′-AACGTTC-3′-HEG-5′-TGACGTT-3′ C-625′-TGACGTT-3′-HEG-5′-AACGTTC-3′-HEG-5′-TCCATTT-3′ C-635′-TCGACTC-3′-HEG-5′-TCGAGCG-3′-HEG-5′-TTCTCTT-3′ C-645′-CTGTGAACGTTCGAGATG-3′ (SEQ ID NO: 83)-HEG-5′-CTGTGAACGTTCGAGATG-3′(SEQ ID NO: 83) C-65 5′-TCGTCGA-3′-HEG-5′-TCGTCGA-3′-HEG-3′-AGCTGCT-5′C-66 5′-TCGTCGAACGTTCGAGATG-3′(SEQ ID NO: 41)-HEG-5′-TCGTCGAACGTTCGAGATG-3′ (SEQ ID NO: 41) C-675′-TCGTCGAACGTTCGAGATG-3′ (SEQ ID NO: 41)-HEG-3′-GTAGAGCTTGCAAGCTGCT-5′(SEQ ID NO: 41) C-68 5′-TCG-3′-HEG-5′-T-3′ C-695′-TCGAT-3′-HEG-5′-TCGAT-3′-HEG-5′-TCGAT-3′-HEG-5′-TCGAT-3′ C-705′-TCGTCGA-3′-HEG-5′-TCGTCGA-3′-HEG-5′-AACGTTC-3′-HEG-5′-AGAT-3′ C-715′-TCGACGT-3′-HEG-5′-TCGACGT-3′-HEG-5′-TCGACGT-3′-HEG-5′-TCGACGT-3′ C-725′-TCCTCCA-3′-HEG-5′-ACCTTAG-3′-HEG-5′-AGATGAT-3′ (no CG) C-735′-ACGTCGA-3′-HEG-5′-ACGTCGA-3′-HEG-5′-ACGTCGA-3′ C-745′-TCGATTT-3′-HEG-5′-TCGATTT-3′-HEG-5′-TCGATTT-3′ C-755′-TTCGATT-3′-HEG-5′-TTCGATT-3′-HEG-5′-TTCGATT-3′ C-765′-TTTCGAT-3′-HEG-5′-TTTCGAT-3′-HEG-5′-TTTCGAT-3′ C-775′-TTTTCGA-3′-HEG-5′-TTTTCGA-3′-HEG-5′-TTTTCGA-3′ C-785′-TCGCTTT-3′-HEG-5′-TCGCTTT-3′-HEG-5′-TCGCTTT-3′ C-795′-TCGGTTT-3′-HEG-5′-TCGGTTT-3′-HEG-5′-TCGGTTT-3′ C-805′-ACGATTT-3′-HEG-5′-ACGATTT-3′-HEG-5′-ACGATTT-3′ C-815′-ATCGAT-3′-HEG-5′-ATCGAT-3′-HEG-5′-ATCGAT-3′ C-825′-ATCGATT-3′-HEG-5′-ATCGATT-3′-HEG-5′-ATCGATT-3′ C-835′-AACGTT-3′-HEG-5′-AACGTT-3′-HEG-5′-AACGTT-3′ C-845′-GsGs-3′-C3-5′-TGC-3′-C3-5′-ATCGAT-3′-C3-5′-GCA-3′-C3-5′-GGsGsGsGsG-3′(s = phosphorothioate linkages, otherwise linkages are phosphodiester)C-85 5′-GsGs-3′-C3-5′-TCGTGC-3′-C3-5′-ATCGAT-3′-C3-5′-GCACGA-3′-C3-5′-GGsGsGsGsG-3′ (s = phosphorothioate linkages, otherwise linkages are phosphodiester) C-86 5′-TGCTGCA-3′-C3-5′-AGCTTGC-3′-C3-5′-AGATGAT-3′(No CG) C-87 5′-GsGsGsGs-3′-C3-5′-ATCGAT-3′-C3-5′-TGATGCATCA-3′-C3-5′-ATCGAT-3′-C3-5′-GsGsGsGsGsG-3′ (s = phosphorothioate linkages,otherwise linkages are phosphodiester) (TGATGCATCA is SEQ ID NO: 105)C-88 5′-TCCA-3′-C3-5′-TGACGTT-3′-C3-5′-CCTGATGCT-3′ C-895′-TGACTGTGA-3′-C3-5′-ACGTTCG-3′-C3-AGATGAT-3′ C-905′-TCGTCGA-3′-C3-5′-TCGTCGA-3′-C3-5′-TCGTCGA-3′ C-915′-TCG-3′-(ab)₃-5′-T-3′ C-92 (ab)-5′-TCG-3′-(ab)₂-5′-T-3′ C-93(5′-TCGTCGA-3′-HEG)₂-glycerol-HEG-5′-TCGTCGA-3′ (phosphodiester) C-94(5′-TCGTCGA-3′-HEG)₂-glycerol-HEG-5′-TCGTCGA-3′ C-95(5′-TCGTCGA-3′-HEG)₂-glycerol-HEG-3′-AGCTGCT-5′ C-96(5′-TCGTCGA-3′-HEG)₂-glycerol-HEG-5′-AACGTTC-3′ C-97(5′-TCGTCGA-3′-HEG)₂-glycerol-HEG-5′-AACGTTC-3′-HEG-5′-TCGA-3′ C-98(5′-TCGTCGA-3′-HEG)₃-trebler-HEG-5′-AACGTTC-3′-HEG-5′-TCGA-3′ C-99TMEA-(5′-TGACTGTGAACGTTCGAGATGA-3′)₃ (SEQ ID NO: 139)(See Ex. 23) C-100(5′-TCGTCGA-3′-HEG)₂-glycerol-HEG-5′-AACGTTC-3′-HEG-5′-TCGACGT-3′ C-101(5′-TCGACGT-3′-HEG)₂-glycerol-HEG-5′-TCGACGT-3′ C-102Starburst Dendrimer ® (See Ex. 24) C-103(5′-TCGTCGA-3′-TEG)₂-glycerol-TEG-5′-TCGTCGA-3′ C-104(5′-TCGTCGA-3′-C3)₂-glycerol-C3-5′-TCGTCGA-3′ C-105TMEA-(S—(CH₂)₃-3′-TAGTAGA-5′-HEG-3′-GCTTGCA-5′-HEG-3′-AGCTGCT-5′)₃(See Ex. 23)C-106 HO(CH₂)₆SS(CH₂)₆-5′-TGACTGTGAACGTTCGAGATGA-3′ (SEQ ID NO: 134)C-107 HS(CH₂)₆-5′-TGACTGTGAACGTTCGAGATGA-3′ (SEQ ID NO: 135) C-110HO(CH₂)₆SS(CH₂)₆-5′-TCGTCG-3′-C3-5′-ACGTTCG-3′-C3-5′-AGATGAT-3′ C-111HS(CH₂)₆-5′-TCGTCG-3′-C3-5′-ACGTTCG-3′-C3-5′-AGATGAT-3′ C-112HO(CH₂)₆SS(CH₂)₆-5′-TCGTCGA-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′ C-113HS(CH₂)₆-5′-TCGTCGA-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′ C-1145′-TCGTCGA-3′-C3-5′-ACGTTCG-3′-C3-5′-AGATGAT-3′-C3-(CH₂)₆SS(CH₂)₆₃OHC-115 5′-TCGTCGA-3′-C3-5′-ACGTTCG-3′-C3-5′-AGATGAT-3′-C3-(CH₂)₃SH C-1165′-TCGTCGA-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′-(CH₂)₃SS(CH₂)₃OH C-1175′-TCGTCGA-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′-(CH₂)₃SH C-1185′-TCGTCGA-3′-HEG-C3-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′ C-1195′-TCGA-3′-HEG-5′-TCGA-3′-HEG-5′-TCGA-3′-HED-5′-TCGA-3′-HEG-5′-TCGA-3′C-120 5′-TCGTCG-3′-C6-5′-ACGTTCG-3′-C6-5′-AGATGAT-3′ C-121(5′-AACGTT-3′-HEG)₂-glycerol-HEG-5′-AACGTT-3′ C-122(5′-TCAACGTT-3′-HEG)₂-glycerol-HEG-5′-TCAACGTT-3′ C-123(5′-TCGTCGA-3′-HEG-HEG)₂-glycerol-HEG-HEG-5′-TCGTCGA-3′ C-124(5′-TCGACGT-3′-HEG)₂-symmetrical doubler-HEG-5′-TCGACGT-3′ C-125(5′-TCGACGT-3′-HEG)₃-trebler-HEG-5′-TCGACGT-3′ C-126((5′-TCGACGT-3′-HEG)₂-glycerol-HEG)₂-glycerol-HEG-5′-TCGACGT-3′ C-127(5′-TCGACGT-3′-HEG)₂-glycerol-HEG-5′-AACGTTC-3′ C-128HO(CH₂)₆SS(CH₂)₆-5′-TCGTCGA-3′-C3-5′-ACGTTCG-3′-C3-5′-AGATGAT-3′ C-129((5′-TCGACGT-3′-HEG)2-glycerol-HEG)2-glycerol-HEG-5′-T-3′ C-130(5′-TCGACGT-3′-HEG)3-trebler-HEG-5′-T-3′ C-1315′-TCGTCGA-3′-C4-5′-ACGTTCG-3′-C4-5′-AGATGAT-3′ C-1325′-TCGTCGA-3′-C6-5′-ACGTTCG-3′-C6-5′-AGATGAT-3′ C-1335′-TCGTCGA-3′-TEG-5′-ACGTTCG-3′-TEG-5′-AGATGAT-3′ C-1345′-TCGTCGA-3′-PEG-5′-ACGTTCG-3′-PEG-5′-AGATGAT-3′[PEG = (CH2CH2O)₄₅]C-135 5′-TCGACGT-3′-HEG-(CH₂)₃SS(CH₂)₃OH C-1365′-TCGACGT-3′-HEG-(CH₂)₃SH C-137(5′-TCGACGT-3′-HEG)_(x)-Ficoll₄₀₀ (X range =150-250, ave. 185) (See example 49) C-138 Ficoll-(5′-P-6)₁₅₆ C-139bPEG-(5′-P-6)₂₋₄ C-140 (5′-TCGACGT-3′-HEG)₂₋₄-bPEG C-141(5′-TCGACGT-3′-HEG)₃-TMEA C-142(5′-TCGTCGT-3′-HEG)₂-glycerol-HEG-5′-TCGTCGT-3′ C-143(5′-TCGTTTT-3′-HEG)₂-glycerol-HEG-5′-TCGTTTT-3′ C-144(5′-TCGAACG-3′-HEG)₂-glycerol-HEG-5′-TCGAACG-3′ C-145(5′-TCGATCG-3′-HEG)₂-glycerol-HEG-5′-TCGATCG-3′ C-146(5′-TCGAGAT-3′-HEG)₂-glycerol-HEG-5′-TCGAGAT-3′ C-147(5′-TCGATTT-3′-HEG)₂-glycerol-HEG-5′-TCGATTT-3′ C-148(5′-TCGACGA-3′-HEG)₂-glycerol-HEG-5′-TCGACGA-3′ C-149(5′-TCGAGCT-3′-HEG)₂-glycerol-HEG-5′-TCGAGCT-3′ C-150(5′-TCGAATT-3′-HEG)₂-glycerol-HEG-5′-TCGAATT-3′ C-1515′-TCGTCGA-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′-C₆NH₂ C-152(5′-TCGACGT-3′-HEG)₂-glycerol-HEG-5′-TCGACGT-3′-C₆NH₂ C-153(5′-TCGACGT-3′-HEG)₂-glycerol-HEG-5′-TCGTCGA-3′ C-154(5′-TCGTCGA-3′-HEG)₂-glycerol-HEG-5′-TCGACGT-3′ C-155

C-156 (5′-TCGCTCG-3′-HEG)₂-glycerol-HEG-5′-TCGCTCG-3′ C-157(5′-TCGGTCG-3′-HEG)₂-glycerol-HEG-5′-TCGGTCG-3′ C-158(5′-TCGTTCG-3′-HEG)₂-glycerol-HEG-5′-TCGTTCG-3′ C-1595′-TGCGTGTAACGTTACACGCA-3′(SEQ ID NO: 114)-HEG-5′-TGCGTGTAACGTTACACGCA-3′ (SEQ ID NO: 114) C-1605′-TGCGTGTAACGTTACACGCA-3′ (SEQ ID NO: 114)-HEG-5′-TGCGTGTAACGTTACAC-3′(SEQ ID NO: 114) C-161 (5′-CTGAACGTTCAG-3′(SEQ ID NO: 104)-HEG)₂-glycerol-HEG-5′-CTGAACGTTCAG-3′ (SEQ ID NO: 104)C-162 (5′-CTGAACGTTCAG-3′(SEQ ID NO: 104)-HEG)₂-glycerol-HEG-3′-GACTTGCAAGTC-5′ (SEQ ID NO: 104)C-163 (5′-CTGAACGTTCAG (SEQ ID NO: 104)-3′-HEG)₃-trebler-HEG-5′-T-3′C-164 (5′-CTGAACGTTCAG (SEQ ID NO: 104)-3′-HEG)₃-trebler-HEG-5′-T-3′(all phosphodiester) C-165 (5′-TGCGTGTAACGTTACACGCA-3′(SEQ ID NO: 114)-HEG)₂-glycerol-HEG-5′-T-3′ C-166(5′-TGCGTGTAACGTTACACGCA-3′)₂ (SEQ ID NO: 114)-glycerol-HEG-5′-T-3′(all phosphodiester) C-167(5′-TCGACGT-3′-HEG)₂-glycerol-HEG-5′-TTGGCCAAGCTTGGCCAA-3′(SEQ ID NO: 116)C-168 5′-TCGTCGA-3′-HEG-(gly(HEG-3′-TGCAGCT-5′)-HEG)3-5′-TCGAACG-3′C-1695′-TCGTCGA-3′-HEG-(gly(HEG-3′-TGCAGCT-5′)-5′-TTTTT-3′)3-HEG-5′-TCGAACG-3′C-170 (5′-TCGACGT-3′-HEG)₂-glycerol-HEG-glycerol-(HEG-3′-TGCAGCT-5′)₂C-171(5′-TCGACGT-3′-HEG)₂-glycerol-5′-TTTTT-3′-glycerol-(HEG-3′-TGCAGCT-5′)₂C-172 5′-TCGTTCGAACGTTCCGAACGA-3′(SEQ ID NO: 153)-HEG-5′-TCGTTCGAACGTTCGAACGA-3′ (SEQ ID NO: 154) C-173(5′-TCGAACGTTCGA-3′(SEQ ID NO: 155)-HEG)₂-glycerol-HEG-5′-TCGAACGTTCGA-3′ (SEQ ID NO: 155)C-175 (5′-TCGAACGTTCGA-3′ (SEQ ID NO: 155)-HEG)₃-trebler-HEG-5′-T-3′C-176 5′-TCGTTCGAACGTTCCGAACGA-3′(SEQ ID NO: 153)-HEG-5′-TCGTTCGAACGTTCGAA-3′ (SEQ ID NO: 156) C-177(5′-TCGTTCGAACGTTCCGAACGA-3′ (SEQ ID NO: 153)-HEG)₂-glycerol-HEG-5′-T-3′C-178(5′-TCGACGT-HEG)₂-glycerol-HEG-5′-TTGGCCAAGCTTGGCCAA(SEQ ID NO: 116)C-179 5′-TCGTCGA-3′-HEG-5′-TCGTCGA-3′-HEG-5′-ACGTTCG-3′ C-1805′-TCGTCGA-3′-TEG-5′-TCGTCGA-3′-TEG-5′-ACGTTCG-3′ C-1815′-TCGTCGA-3′-C6-5′-TCGTCGA-3′-C6-5′-ACGTTCG-3′ C-1825′-TCGTCGA-3′-HEG-5′-ACGTTCG-3′-HEG-5′-TCGAGAT-3′ C-1835′-TCGACGT-3′-TEG-5′-TCGTCGA-3′-TEG-5′-ACGTTCG-3′ C-1845′-TCGTCGA-3′-TEG-5′-TCGACGT-3′-TEG-5′-ACGTTCG-3′ C-1855′-TCGACGT-3′-HEG-5′-TCGACGT-3′-HEG-5′-TCGTCGA-3′ C-1865′-TCGACGT-3′-HEG-5′-TCGACGT-3′-C3-5′-ACGTTCG-3′ C-1875′-TCGAACGTTCGA-3′(SEQ ID NO: 155)-HEG-5′-TCGAACGTTCGA-3′(SEQ ID NO: 155) C-188(5′-TCGAACGTTCGA-3 (SEQ ID NO: 155)′-HEG)₂-glycerol-HEG-5′-T-3′ C-1895′-TCGACGT-3′-HEG-5′-TCGACGT-3′-HEG-5′-AACGTTC-3′ C-1905′-TCGACGT-3′-HEG-5′-AACGTTC-3′-HEG-5′-TCGACGT-3′ C-1915′-TCGACGT-3′-HEG-5′-TCGACGT-3′-HEG-5′-TCGACGT-3′-HEG-5′-AACGTTC-3′C-1925′-TCGACGT-3′-HEG-5′-TCGACGT-3′-HEG-5′-AACGTTC-3′-HEG-5′-TCGACGT-3′C-193 5′-TCGACGT-3′-HEG-5′-TCGACGT-3′-TEG-5′-ACGTTCG-3′ C-1945′-TCGACGT-3′-HEG-5′-TCGACGT-3′-C6-5′-ACGTTCG-3′ C-1955′-TCGACGT-3′-HEG-5′-TCGACGT-3′-C4-5′-ACGTTCG-3′ C-196(5′-TCGACGT-HEG)₂-glycerol-HEG-5′-TCGACGT-3′-HEG-5′-AACGTTC-3′ C-1975′-TCGATCG-3′-HEG-5′-TCGATCG-3′-HEG-5′-TCGATCG-3′ C-198(5′-TCGGCGC-HEG)₂-glycerol-HEG-5′-TCGGCGC-3′ C-199(5′-TCGCCGG-HEG)₂-glycerol-HEG-5′-TCGCCGG-3′ C-200(5′-TCGACGT-HEG)₂-glycerol-C4-5′-ACGTTCG-3′ C-201(5′-TCGACGT-HEG)₂-glycerol-5′-TCG-3′-C4-5′-TCGACGT-3′ C-202(5′-TCGACGT-HEG)₂-glycerol-HEG-5′-ACTTAGAGGTTCAGTAGG-3′(SEQ ID NO: 157)C-203(5′-TCGACGT-HEG)₂-glycerol-HEG-5′-CCTACTGAACCTCTAAGT-3′(SEQ ID NO: 158)C-204 (5′-AACGTTC-HEG)₂-glycerol-5′-AACGTTC-3′ C-205(5′-TCGACGT-HEG)₂-glycerol-5′-GACGTTC-3′ C-206(5′-TCGACGT-HEG)₂-glycerol-5′-GACGTCC-3′ C-207(5′-TCGACGT-HEG)₂-glycerol-5′-AGCGCTC-3′ C-208(5′-TCGTTCG-HEG)₂-glycerol-HEG-5′-ACTTAGAGGTTCAGTAGG-3′(SEQ ID NO: 157)C-209(5′-TCGTTCG-HEG)₂-glycerol-HEG-5′-CCTACTGAACCTCTAAGT-3′(SEQ ID NO: 158)

Example 2 Synthesis of a Chimeric Compound with a Linear Structure andHexaethylene Glycol Spacers

C-10, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages, and the spacermoieties are hexaethylene glycol (HEG), connected to the nucleic acidmoieties via phosphorothioate linkages.

C-10: 5′-TCGTCG-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′

The C-10 molecule was synthesized by TriLink BioTechnologies (SanDiego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturer's protocol for 1 umolphosphorothioate DNA. The nucleoside monomers and the spacer moietyprecursor, 4,4′-O-dimethoxytrityl-hexaethyleneglycol-O—(N,N-diisopropyl) 2-cyanoethylphosphoramidite (obtained fromGlen Research, Sterling, Va.) were dissolved in anhydrous acetonitrileto a final concentration of 0.05 M. (As will be apparent to theordinarily skilled reader, the terms “nucleoside monomer” or “spacermoiety” are sometimes used herein, e.g., in the context of synthesis ofCICs, to refer to the precursor reagents that when deprotected andlinked to other components using synthetic methods such as thosedisclosed herein, give rise to the nucleic acid and nonnucleic acidmoieties of the CICs.) The HEG spacer precursor was placed in anauxiliary monomer site on the instrument. The instrument was programmedto add the nucleotide monomers and HEG spacers in the desired order,with synthesis of the nucleic acid moieties occurring in the 3′ to 5′direction.

-   -   1. Use a 3′-support bound “T” solid support    -   2. Synthesis of 5′-AGATGA-3′ moiety    -   3. Addition of HEG spacer    -   4. Synthesis of 5′-ACGTTCG-3′ moiety    -   5. Addition of HEG spacer    -   6. Synthesis of 5′-TCGTCG-3′ moiety

The synthesis cycle consisted of a detritylation step, a coupling step(phosphoramidite monomer plus 1H-tetrazole), a capping step, asulfurization step using 0.05 M 3H-1,2-benzodithiol-3-one 1,1-dioxide(Beaucage reagent), and a final capping step. At the completion ofassembly, the ‘trityl-off’ compound was cleaved from the controlled-poreglass and the bases were deprotected with concentrated aqueous ammoniaat 58° C. for 16 hours. The compound was purified by preparativepolyacrylamide electrophoresis, desalted on a Sep-pak Plus cartridge(Waters, Milford, Mass.), and precipitated from 1 M aqueous sodiumchloride with 2.5 volumes of 95% ethanol. The molecule was dissolved inMilli Q water and the yield was determined from the absorbance at 260nm. Finally, the compound was lyophilized to a powder. The compound wascharacterized by capillary gel electrophoresis, electrospray massspectrometry, and RP-HPLC to confirm composition and purity. Anendotoxin content assay (LAL assay, Bio Whittaker) was also conducted,showing endotoxin levels were <5 EU/mg compound (i.e., essentiallyendotoxin free).

C-8,C-21,C-22,C-23,C-24, C-32 and M-1 and other linear HEG-CICswere synthesized analogously.

Example 3 Synthesis of a Chimeric Compound with a Linear Structure andPropyl Spacers

C-11, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages, and the spacermoieties are propyl (C3), connected to the nucleic acid moieties viaphosphorothioate linkages.

C-11: 5′-TCGTCG-3′-C3-5′-ACGTTCG-3′-C3-5′-AGATGAT-3′

The C-11 molecule was synthesized by TriLink BioTechnologies (San Diego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturer's protocol for 1 umolphosphorothioate DNA. The nucleoside monomers and the spacer moietyprecursor, 4,4′-O-dimethoxytrityl-propyl-O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (obtained from Glen Research, Sterling, Va.)were dissolved in anhydrous acetonitrile to a final concentration of0.05 M. The C3 spacer precursor was placed in an auxiliary monomer siteon the instrument. The instrument was programmed to add the nucleotidemonomers and C3 spacers in the desired order, with synthesis of thenucleic acid moieties occurring in the 3′ to 5′ direction.

-   -   1. Use a 3′-support bound “T” solid support    -   2. Synthesis of 5′-AGATGA-3′ moiety    -   3. Addition of C3 spacer    -   4. Synthesis of 5′-ACGTTCG-3′ moiety    -   5. Addition of C3 spacer    -   6. Synthesis of 5′-TCGTCG-3′ moiety

The synthesis, deprotection, workup, and analysis were performed asdescribed in Example 2.

C-9 and other C3-containing CICs were synthesized analogously.

Example 4 Synthesis of a Chimeric Compound with a Linear Structure andwith Butyl Spacers

C-17, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages, and the spacermoieties are butyl (C4), connected to the nucleic acid moieties vianhosnhorothinate linkages.

C-17: 5′-TCGTCG-3′-C4-5′-ACGTTCG-3′-C4-5′-AGATGAT-3′

The C-17 molecule was synthesized by TriLink BioTechnologies (San Diego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturer's protocol for 1 umolphosphorothioate DNA. The nucleoside monomers and the spacer moietyprecursor, 4,4′-O-dimethoxytrityl-butyl-O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (obtained from ChemGenes, Ashland, Mass.)were dissolved in anhydrous acetonitrile to a final concentration of0.05 M. The C4 spacer precursor was placed in an auxiliary monomer siteon the instrument. The instrument was programmed to add the nucleotidemonomers and C4 spacers in the desired order, with synthesis of thenucleic acid moieties occurring in the 3′ to ° 5′ direction.

-   -   1. Use a 3′-support bound “T” solid support    -   2. Synthesis of 5′-AGATGA-3′ moiety    -   3. Addition of C4 spacer    -   4. Synthesis of 5′-ACGTTCG-3′ moiety    -   5. Addition of C4 spacer    -   6. Synthesis of 5′-TCGTCG-3′ moiety

The synthesis, deprotection, workup, and analysis were performed asdescribed in Example 2.

Example 5 Synthesis of a Chimeric Compound with a Linear Structure andTriethylene Glycol Spacers

C-18, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages, and the spacermoieties are triethylene glycol (TEG), connected to the nucleic acidmoieties via phosphorothioate linkages.

C-18: 5′-TCGTCG-3′-TEG-5′-ACGTTCG-3′-TEG-5′-AGATGAT-3′

The C-18 molecule was synthesized by TriLink BioTechnologies (San Diego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturer's protocol for 1 umolphosphorothioate DNA. The nucleoside monomers and the spacer moietyprecursor, 4,4′-O-dimethoxytrityl-triethylene glycol-O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (obtained from Glen Research, Sterling, Va.)were dissolved in anhydrous acetonitrile to a final concentration of0.05 M. The TEG spacer precursor was placed in an auxiliary monomer siteon the instrument. The instrument was programmed to add the nucleotidemonomers and TEG spacers in the desired order, with synthesis of thenucleic acid moieties occurring in the 3′ to 5′ direction.

-   -   1. Use a 3′-support bound “T” solid support    -   2. Synthesis of 5′-AGATGA-3′ moiety    -   3. Addition of TEG spacer    -   4. Synthesis of 5′-ACGTTCG-3′ moiety    -   5. Addition of TEG spacer    -   6. Synthesis of 5′-TCGTCG-3′ moiety

The synthesis, deprotection, workup, and analysis were performed asdescribed in Example 2.

Example 6 Synthesis of a Chimeric Compound with a Linear Structure andDodecyl Spacers

C-19, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages, and the spacermoieties are dodecyl (C12), connected to the nucleic acid moieties viaphosphorothioate linkages.

C-19: 5′-TCGTCG-3′-C12-5′-ACGTTCG-3′-C12-5′-AGATGAT-3′

The C-19 molecule was synthesized by TriLink BioTechnologies (San Diego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturer's protocol for 1 umolphosphorothioate DNA. The nucleoside monomers and the spacer moietyprecursor, 4,4′-O-dimethoxytrityl-dodecyl-O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (obtained from Glen Research, Sterling, Va.)were dissolved in anhydrous acetonitrile to a final concentration of0.05 M. The C12 spacer precursor was placed in an auxiliary monomer siteon the instrument. The instrument was programmed to add the nucleotidemonomers and C12 spacers in the desired order, with synthesis of thenucleic acid moieties occurring in the 3′ to 5′ direction.

-   -   1. Use a 3′-support bound “T” solid support    -   2. Synthesis of 5′-AGATGA-3′ moiety    -   3. Addition of C12 spacer    -   4. Synthesis of 5′-ACGTTCG-3′ moiety    -   5. Addition of C12 spacer    -   6. Synthesis of 5′-TCGTCG-3′ moiety

The synthesis, deprotection, workup, and analysis were performed asdescribed in Example 2.

Example 7 Synthesis of a Chimeric Compound with a Linear Structure andAbasic Spacers

C-20, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages, and the spacermoieties are 1′,2′-dideoxyribose (abasic), connected to the nucleic acidmoieties via phosphorothioate linkages.

C-20: 5′-TCGTCG-3′-abasic-5′-ACGTTCG-3′-abasic-5′- AGATGAT-3′

The C-20 molecule was synthesized by TriLink BioTechnologies (San Diego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturer's protocol for 1 umolphosphorothioate DNA. The nucleoside monomers and the spacer moietyprecursor,5′-O-(4,4′-dimethoxytrityl)-1′,2′-dideoxyribose-3′-O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (obtained from Glen Research, Sterling, Va.)were dissolved in anhydrous acetonitrile to a final concentration of0.05 M. The abasic spacer precursor was placed in an auxiliary monomersite on the instrument. The instrument was programmed to add thenucleotide monomers and abasic spacers in the desired order, withsynthesis of the nucleic acid moieties occurring in the 3′ to 5′direction.

-   -   1. Use a 3′-support bound “T” solid support    -   2. Synthesis of 5′-AGATGA-3′ moiety    -   3. Addition of abasic spacer    -   4. Synthesis of 5′-ACGTTCG-3′ moiety    -   5. Addition of abasic spacer    -   6. Synthesis of 5′-TCGTCG-3′ moiety

The synthesis, deprotection, workup, and analysis were performed asdescribed in Example 2.

Example 8 Synthesis of a Chimeric Compound with a Linear Structure andHexaethylene Glycol and Triethylene Glycol Spacers

C-29, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages, the spacermoieties are hexaethylene glycol (HEG), connected to the nucleic acidmoieties via phosphorothioate linkages, and the 3′-end group istriethylene glycol (TEG), connected to the nucleic acid moiety via aphosphorothioate linkage.

C-29: 5′-TCGTCG-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT- 3′-TEG

The C-29 molecule was synthesized by TriLink BioTechnologies (San Diego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturer's protocol for 1 umolphosphorothioate DNA. The triethylene glycol-controlled-pore glass, usedas the solid support for the synthesis, was from Glen Research(Sterling, Va.). The nucleoside monomers and the spacer moietyprecursor, 4,4′-O-dimethoxytrityl-hexaethyleneglycol-O—(N,N-diisopropyl) 2-cyanoethylphosphoramidite (obtained fromGlen Research, Sterling, Va.) were dissolved in anhydrous acetonitrileto a final concentration of 0.05 M. The REG spacer was placed in anauxiliary monomer site on the instrument. The instrument was programmedto add the nucleotide monomers and HEG spacers in the desired order,with synthesis of the nucleic acid moieties occurring in the 3′ to 5′direction.

-   -   1. Use a triethylene glycol solid support    -   2. Synthesis of 5′-AGATGAT-3′ moiety    -   3. Addition of HEG spacer    -   4. Synthesis of 5′-ACGTTCG-3′ moiety    -   5. Addition of HEG spacer

6. Synthesis of 5′-TCGTCG-3′ moiety

The synthesis, deprotection, workup, and analysis were performed asdescribed in Example 2.

Example 9 Synthesis of a Chimeric Compound with a Linear Structure andHexaethylene Glycol and Triethylene Glycol Spacers

C-30, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages, the spacermoieties and 5′-end group are hexaethylene glycol (HEG), connected tothe nucleic acid moieties via phosphorothioate linkages, and the 3′-endgroup is triethylene glycol (TEG), connected to the nucleic acid moietyvia a phosphorothioate linkage.

C-30: HEG-5′-TCGTCG-3′-HEG-5′-ACGTTCG-3′-HEG-5′- AGATGAT-3′-TEG

The C-30 molecule was synthesized by TriLink BioTechnologies (San Diego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturer's protocol for 1 umolphosphorothioate DNA. The triethylene glycol-controlled-pore glass, usedas the solid support for the synthesis, was from Glen Research(Sterling, Va.). The nucleoside monomers and the spacer moietyprecursor, 4,4′-O-dimethoxytrityl-hexaethyleneglycol-O—(N,N-diisopropyl) 2-cyanoethylphosphor amidite (obtained fromGlen Research, Sterling, Va.) were dissolved in anhydrous acetonitrileto a final concentration of 0.05 M. The HEG spacer precursor was placedin an auxiliary monomer site on the instrument. The instrument wasprogrammed to add the nucleotide monomers and HEG spacers in the desiredorder, with synthesis of the nucleic acid moieties occurring in the 3′to 5′ direction.

-   -   1. Use a triethylene glycol solid support    -   2. Synthesis of 5′-AGATGAT-3′ moiety    -   3. Addition of HEG spacer    -   4. Synthesis of 5′-ACGTTCG-3′ moiety    -   5. Addition of HEG spacer    -   6. Synthesis of 5′-TCGTCG-3′ moiety    -   7. Addition of the HEG spacer

The synthesis, deprotection, workup, and analysis were performed asdescribed in Example 2.

Example 10 Synthesis of a Chimeric Compound with a Linear Structure andHexaethylene Glycol and Triethylene Glycol Spacers, and withPhosphodiester Linkages

C-31, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphodiester linkages, the spacer moietiesand 5′-end group are hexaethylene glycol (HEG), connected to the nucleicacid moieties via phosphodiester linkages, and the 3′-end group istriethylene glycol (TEG), connected to the nucleic acid moiety via aphosphodiester linkage.

C-31: HEG-5′-TCGTCG-3′-HEG-5′-ACGTTCG-3′-HEG-5′- AGATGAT-3′-TEG

The C-31 molecule was synthesized by TriLink BioTechnologies (San Diego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturer's protocol for 1 umol phosphodiesterDNA. The triethylene glycol-controlled-pore glass, used as the solidsupport for the synthesis, was from Glen Research (Sterling, Va.). Thenucleoside monomers and the spacer moiety,4,4′-O-dimethoxytrityl-hexaethylene glycol-O—(N,N-diisopropyl)2-cyanoethylphosphor amidite (obtained from Glen Research, Sterling,Va.) were dissolved in anhydrous acetonitrile to a final concentrationof 0.05 M. The HEG spacer was placed in an auxiliary monomer site on theinstrument. The instrument was programmed to add the nucleotide monomersand HEG spacers in the desired order, with synthesis of the nucleic acidmoieties occurring in the 3′ to 5′ direction.

-   -   1. Use a triethylene glycol solid support    -   2. Synthesis of 5′-AGATGAT-3′ moiety    -   3. Addition of HEG spacer    -   4. Synthesis of 5′-ACGTTCG-3′ moiety    -   5. Addition of HEG spacer    -   6. Synthesis of 5′-TCGTCG-3′ moiety    -   7. Addition of the HEG spacer

The synthesis cycle consisted of a detritylation step, a coupling step(phosphoramidite monomer plus 1H-tetrazole), a capping step, anoxidation step, and a final capping step. At the completion of assembly,the ‘trityl-off’ compound was cleaved from the controlled-pore glass andthe bases were deprotected with concentrated aqueous ammonia at 58° C.for 16 hours. The compound was purified by preparative polyacrylamideelectrophoresis, desalted on a Sep-pak Plus cartridge (Waters, Milford,Mass.), and precipitated from 1 M aqueous sodium chloride with 2.5volumes of 95% ethanol. The compound was dissolved in Milli Q water andthe yield was determined from the absorbance at 260 nm. Finally, thecompound was lyophilized to a powder. The compound was characterized bycapillary gel electrophoresis, electrospray mass spectrometry, andRP-HPLC to confirm composition and purity. An endotoxin content assay(LAL assay, Bio Whittaker) was also conducted, showing endotoxin levelswere <5 EU/mg compound.

Example 11 Synthesis of a Chimeric Compound with a Linear Structure and2-(Hydroxymethyl)ethyl Spacers

C-25, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages, and the spacermoieties are 2-(hydroxymethyl)ethyl (HME), connected to the nucleic acidmoieties via phosphorothioate linkages.

C-25: 5′-TCGTCG-3′-HME-5′-ACGTTCG-3′-HME-5′-AGATGAT-3′

The C-25 molecule was synthesized by TriLink BioTechnologies (San Diego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturer's protocol for 1 umolphosphorothioate DNA. The nucleoside monomers and the spacer moietyprecursor,1-O-(4,4′-dimethoxytrityl)-3-β-levulinyl-glycerol-2-O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (obtained from ChemGenes, Ashland, Mass.)were dissolved in anhydrous acetonitrile to a final concentration of0.05 M. The HME spacer was placed in an auxiliary monomer site on theinstrument. The instrument was programmed to add the nucleotide monomersand HME spacers in the desired order, with synthesis of the nucleic acidmoieties occurring in the 3′ to 5′ direction.

-   -   1. Use a 3′-support bound “T” solid support    -   2. Synthesis of 5′-AGATGA-3′ moiety    -   3. Addition of TIME spacer    -   4. Synthesis of 5′-ACGTTCG-3′ moiety    -   5. Addition of HME spacer    -   6. Synthesis of 5′-TCGTCG-3′ moiety

The synthesis, deprotection, workup, and analysis were performed asdescribed in Example 2. The levulinyl group is removed during thetreatment with ammonia.

Example 12 Synthesis of a Chimeric Compound with a Linear Structure anda Negatively Charged Spacer Moiety

C-13, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages, and the spacermoiety is a propyl (C3) polymer linked via phosphorothioate linkages.

C-13: 5′-TCGTCG-3′-(C3)₁₅-5′-T-3′

The C-13 molecule was synthesized on a Perseptive Biosystems Expedite8909 automated DNA synthesizer using the manufacturers protocol for 1umol phosphorothioate DNA. The nucleoside monomers and the spacer moietyprecursor, 4,4′-O-dimethoxytrityl-propyl-O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (obtained from Glen Research, Sterling, Va.)were dissolved in anhydrous acetonitrile to a final concentration of 0.1M. The C3 spacer was placed in an auxiliary monomer site on theinstrument. The instrument was programmed to add the nucleotide monomersand C3 spacers in the desired order, with synthesis of the nucleic acidmoieties occurring in the 3′ to 5′ direction.

-   -   1. Use a 3′-support bound “T” solid support    -   2. Addition of 15 C3 spacers    -   3. Synthesis of 5′-TCGTCG-3′ moiety

The synthesis cycle consisted of a detritylation step, a coupling step(phosphoramidite monomer plus 1H-tetrazole), a capping step, asulfurization step using 0.02 M 3-amino-1,2,4-dithiazole-5-thione (ADTT)in 9:1 acetonitrile:pyridine, and a final capping step. At thecompletion of assembly, the ‘trityl-on’ compound was cleaved from thecontrolled-pore glass and the bases were deprotected with concentratedaqueous ammonia at 58° C. for 16 hours. The compound was purified byHPLC on a Hamilton PRP-1 column using an increasing gradient ofacetonitrile in 0.1 M triethylammonium acetate. The purified compoundwas concentrated to dryness, the 4,4′-dimethoxytrityl group was removedwith 80% aqueous acetic acid, and then the compound was precipitated twotimes from 1 M aqueous sodium chloride with 2.5 volumes of 95% ethanol.The compound was dissolved in Milli Q water and the yield was determinedfrom the absorbance at 260 nm. Finally, the compound was lyophilized toa powder.

The compound was characterized by capillary gel electrophoresis,electrospray mass spectrometry, and RP-HPLC to confirm composition andpurity. An endotoxin content assay (LAL assay, Bio Whittaker) was alsoconducted, showing endotoxin levels were <5 EU/mg compound.

C-14, C-15 and C-16 were synthesized analogously.

Example 13 Synthesis of a Chimeric Compound with a Linear Structure anda Negatively Charged Spacer Moiety

C-38, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages, and the spacermoieties are hexaethylene glycol (HEG), connected via phosphorothioatelinkages.

C-38: 5′-TCGTCGA-3′-(HEG)₄-5′-TCGTCGA-3′

The C-38 molecule was synthesized as described in Example 2. The spacermoiety precursor is 4,4′-O-dimethoxytrityl-hexaethyleneglycol-O—(N,N-diisopropyl) 2-cyanoethylphosphoramidite (obtained fromGlen Research, Sterling, Va.). The synthesis was accomplished bycarrying out the following steps:

-   -   1. Use a 3′-support bound “A” solid support    -   2. Synthesis of 5′-TCGTCG-3′ moiety    -   3. Addition of 4 HEG spacers    -   4. Synthesis of 5′-TCGTCGA-3′ moiety

The compound was purified using HPLC as described in Example 12. Thecompound was characterized and the endotoxin content determined asdescribed in Example 2.

Example 14 Synthesis of a Chimeric Compound with a Linear Structure anda Negatively Charged Spacer Moiety with Both Nucleic Acid MoietiesAttached Via the 3′-End

C-37, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages, and the spacermoieties are hexaethylene glycol (HEG), connected via phosphorothioatelinkages.

C-37: 5′-TCGTCGA-3′-(HEG)-3′-AGCTGCT-5′

The C-37 molecule was synthesized as described in Example 2, except thata 5′-support bound nucleoside and 3′-O-(4,4′-dimethoxytrityl)-protectednucleoside-5′-O—(N,N-diisopropyl) 2-cyanoethylphosphoramidites were used(Glen Research, Sterling, Va.) to synthesize the first nucleic acidmoiety. The spacer moiety precursor is4,4′-O-dimethoxytrityl-hexaethylene glycol-O—(N,N-diisopropyl)2-cyanoethylphosphor amidite (obtained from Glen Research, Sterling,Va.). The synthesis was accomplished by carrying out the followingsteps:

-   -   1. Use a 5′-support bound “T” solid support    -   2. Synthesis of 3′-AGCTGC-5′ moiety with        3′-O-(4,4′-dimethoxytrityl)-protected        nucleoside-5′-O—(N,N-diisopropyl) 2-cyanoethylphosphoramidites        (5′ to 3′ synthesis)    -   3. Addition of 4 HEG spacers    -   4. Synthesis of 5′-TCGTCGA-3′ moiety with        5′-O-(4,4′-dimethoxytrityl)-protected        nucleoside-3′-O—(N,N-diisopropyl) 2-cyanoethylphosphoramidites        (3′ to 5′ synthesis)

The compound was purified using HPLC as described in Example 12. Thecompound was characterized and the endotoxin content determined asdescribed in Example 2.

Example 15 Synthesis of a Chimeric Compound with a Branched Structure

C-27, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages and the spacermoiety is glycerol, connected to the nucleic acid moieties viaphosphorothioate linkages.

C-27:(5′-TCGTCGA-3′)₂-glycerol-5′-AACGTTC-3′

The C-27 molecule was synthesized by TriLink BioTechnologies (SanDiego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturers protocol for 1 umol phosphorothioateDNA. The nucleoside monomers and the spacer moiety precursor,1,3-di-(4,4′-β-dimethoxytrityl)-glycerol-2-O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (symmetrical branched phosphoramiditeobtained from ChemGenes, Ashland, Mass., FIG. 2) were dissolved inanhydrous acetonitrile to a final concentration of 0.05 M. The glycerolspacer was placed in an auxiliary monomer site on the instrument. Theinstrument was programmed to add the nucleotide monomers and theglycerol spacer in the desired order, with synthesis of the nucleic acidmoieties occurring in the 3′ to 5′ direction.

-   -   1. Use a 3′-support bound “C” solid support    -   2. Synthesis of 5′-AACGTT-3′ moiety    -   3. Addition of the symmetrical branched phosphoramidite based on        glycerol    -   4. Synthesis of two 5′-TCGTCGA-3′ moieties simultaneously

The preparation of this branched compound followed the same protocoldescribed in Example 2, except that in step 4, each reagent delivery inthe synthesis cycle was doubled because two nucleic acid chains werebuilt simultaneously. The symmetrical branched phosphoramidite shown inFIG. 2 requires the nucleic acid sequences synthesized after theaddition of the symmetrical branched phosphoramidite to be the same,although the nucleic acid sequence synthesized before its addition maybe the same or different from the later sequences.

The branched compound was purified and characterized as described inExample 2.

C-28 was synthesized analogously.

Example 16 Synthesis of a Chimeric Compound with a Branched Structureand with all Nucleic Acid Moieties Attached via the 3′-end

C-95, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages and the spacermoieties are glycerol and HEG, connected to the nucleic acid moietiesvia phosphorothioate linkages.

C-95: (5′-TCGTCGA-3′-HEG)₂-glycerol-HEG-3′- AGCTGCT-5′

The C-95 molecule was synthesized as described in Example 2, except thata 5′-support bound nucleoside and 3′-O-(4,4′-dimethoxytrityl)-protectednucleoside-5′-O—(N,N-diisopropyl) 2-cyanoethylphosphoramidites were used(Glen Research, Sterling, Va.) to synthesize the first nucleic acidmoiety. The branched spacer moiety precursor is1,3-di-(4,4′-O-dimethoxytrityl)-glycerol-2-O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (symmetrical branched phosphoramiditeobtained from ChemGenes, Ashland, Mass., FIG. 2). The synthesis wasaccomplished by carrying out the following steps:

-   -   1. Use a 5′-support bound “T” solid support    -   2. Synthesis of 3′-AGCTGC-5′ moiety with        3′-O-(4,4′-dimethoxytrityl)-protected        nucleoside-5′-O—(N,N-diisopropyl) 2-cyanoethyl phosphoramidites        (5′ to 3′ synthesis)    -   3. Addition of a HEG spacer    -   4. Addition of the symmetrical branched phosphoramidite based on        glycerol    -   5. Addition of two HEG spacers simultaneously    -   6. Synthesis of two 5′-TCGTCGA-3′ moieties simultaneously with        5′-O-(4,4′-dimethoxytrityl)-protected        nucleoside-3′-O—(N,N-diisopropyl) 2-cyanoethylphosphoramidites        (3′ to 5′ synthesis)

The preparation of this branched compound followed the same protocoldescribed in Example 2, except that in steps 5 and 6, each reagentdelivery in the synthesis cycle was doubled because two nucleic acidchains were built simultaneously. The symmetrical branchedphosphoramidite shown in FIG. 2 requires the nucleic acid sequencessynthesized after the addition of the symmetrical branchedphosphoramidite to be the same, although the nucleic acid sequencesynthesized before its addition may be the same or different from thelater sequences.

The compound was purified using HPLC as described in Example 12. Thecompound was characterized and the endotoxin content determined asdescribed in Example 2.

Example 17 Synthesis of a Chimeric Compound with a Branched Structure,Containing Three Different Nucleic Acid Moieties

C-35, having the formula shown below, is synthesized. The nucleic acidmoieties are DNA with phosphorothioate linkages and the spacer moiety isglycerol, connected to the nucleic acid moieties via phosphorothioatelinkages.

The C-35 molecule is synthesized as described in Example 2. Thenucleoside monomers and the spacer moiety precursor,1-(4,4′-O-dimethoxytrityl)-3-0-levulinyl-glycerol-2-O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (asymmetrical branched phosphoramiditeobtained from ChemGenes, Ashland, Mass., FIG. 2) are dissolved inanhydrous acetonitrile to a final concentration of 0.05 M. The glycerolspacer is placed in an auxiliary monomer site on the instrument. Theinstrument is programmed to add the nucleotide monomers and the glycerolspacer in the desired order, with synthesis of the nucleic acid moietiesoccurring in the 3′ to 5′ direction.

-   -   1. Use a 3′-support bound “T” solid support    -   2. Synthesis of 5′-AGATGA-3′ moiety    -   3. Addition of the asymmetrical branched phosphoramidite based        on glycerol    -   4. Synthesis of the 5′-AACGTTC-3′ moiety at the dimethoxytrityl        end    -   5. Detritylation and capping of the AACGTTC moiety    -   6. Removal of the levulinyl protecting group    -   7. Synthesis of the 5′-TCGTCGA-3′ moiety

Synthesis takes place essentially as described in Example 2, except thatafter step 4, the 5′-AACGTTC-3′ moiety is detritylated and capped withacetic anhydride/N-methylimidazole in order to terminate that nucleicacid moiety. Next, the levulinyl protecting group is removed with 0.5 Mhydrazine hydrate in 3:2 pyridine:acetic acid/pH 5.1 for 5 min. Thecompound-containing solid support is washed well with anhydrousacetonitrile, and the 5′-TCGTCGA-3′ moiety is added using the protocoldescribed in Example 2.

The branched compound is purified and characterized as described inExample 2.

Example 18 Synthesis of a Chimeric Compound with a Branched Structure bya Conjugation Strategy

C-36 is synthesized as shown in FIG. 3. The nucleic acid moieties areDNA with phosphonothioate linkages and the spacer moiety is based on aSTARBURST® dendrimer. The nucleic acid moiety is synthesized with a5′-C6-disulfide spacer (thiol-modifier C6 S—S, Glen Research, Sterling,Va. product no. 10-1926-xx), which upon reduction, provides a thiolgroup that can react with the maleimide groups on the dendrimer.

Synthesis of 5′-C6-disulfide-TCGTCGA (4)

The 5′-C6-disulfide-TCGTCGA is synthesized using a Perseptive BiosystemsExpedite 8909 automated DNA synthesizer using the manufacturer'sprotocol for 1 umol phosphorothioate DNA. The nucleoside monomers andthe thiol-modifier C6 S—S (Glen Research, Sterling, Va.) are dissolvedin anhydrous acetonitrile to a final concentration of 0.1 M. Thethio-modifier is placed in an auxiliary monomer site on the instrument.The instrument is programmed to add the nucleotide monomers and thethiol modifier in the desired order, with synthesis of the nucleic acidmoieties occurring in the 3′ to 5′ direction.

-   -   1. Use a 3′-support bound “A” solid support    -   2. Synthesis of 5′-TCGTCG-3′ moiety    -   3. Addition of the thiol modifier precursor        (S-trityl-6-mercaptohexyl)-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite)

The synthesis cycle consists of a detritylation step, a coupling step(phosphoramidite monomer plus 1H-tetrazole), a capping step, asulfurization step using 0.02 M 3-amino-1,2,4-dithiazole-5-thione (ADTT)in 9:1 acetonitrile:pyridine, and a final capping step. At thecompletion of assembly, the ‘trityl-on’ compound is cleaved from thecontrolled-pore glass and the bases are deprotected with concentratedaqueous ammonia at 58° C. for 16 hours. The compound is purified by HPLCon a Hamilton PRP-1 column using an increasing gradient of acetonitrilein 0.1 M triethylammonium acetate. The purified compound is concentratedto dryness, the 4,4′-dimethoxytrityl group is removed with 80% aqueousacetic acid, and then the compound is precipitated two times from 1 Maqueous sodium chloride with 2.5 volumes of 95% ethanol. The compound isdissolved in Milli Q water and the yield is determined from theabsorbance at 260 nm. Finally, the compound is lyophilized to a powder.

The compound is characterized by capillary gel electrophoresis,electrospray mass spectrometry, and RP-HPLC to confirm composition andpurity. An endotoxin content assay (LAL assay, Bio Whittaker) is alsoconducted, showing endotoxin levels were <5 EU/mg compound.

Synthesis of 5′-thiol-C6-TCGTCGA (5)

The disulfide modified nucleic acid (4) is reduced to a thiol usingtris(2-carboxyethylphosphine) hydrochloride (TCEP; Pierce, Rockford,Ill.). The nucleic acid is dissolved at a concentration of 20 mg/ml inbuffer containing 0.1 M sodium phosphate/0.15 M sodium chloride/pH 7.5.In a separate vial, the TCEP is dissolved to a concentration of 0.17 Min 0.1 M sodium phosphate/0.15 M sodium chloride/pH 7.5. Add 5equivalents of TCEP to the nucleic acid and mix gently. Incubate thesolution for 120 min at 40° C. and then purify by size exclusionchromatography (Pharmacia P2 column) to yield the 5′-thiol-C6-TCGTCGA(5).

Synthesis of the maleimide-modified STARBURST® dendrimer (7)

STARBURST® dendrimers with various numbers of amines (4, 8, 16, 32, 64,etc.) are available from Aldrich (Milwaukee, Wis.). A Starburst®dendrimer (6), having four amino groups, is dissolved indimethylformamide (DMF) at a concentration of 0.2 M. Triethylamine (10equivalents) and sulfosuccinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC; Pierce,Rockford, Ill., 8 equivalents) are then added and the solution isstirred for 2 hours or until complete, as determined by thin layerchromatography (TLC; 10% methanol/dichloromethane). The reaction isquenched with water for 30 min and then the DMF is removed in vacuo. Theresidue is dissolved in dichloromethane and washed two times withaqueous saturated sodium bicarbonate and then water. The organic phaseis dried over MgSO₄, filtered, and concentrated to dryness in vacuo. Theproduct is purified by silica gel chromatography to yield 7.

Synthesis of STARBURST® dendrimer-(5′-TCGTCGA-3′)₄ (8)

The maleimide-modified STARBURST® dendrimer (6) is dissolved in DMSO (5mg/ml) and the purified 5′-C6-thiol-TCGTCGA (5) (10 equivalents),dissolved at a concentration of 10 mg/ml in 0.1 M sodium phosphate/0.15M sodium chloride/pH 7.5, is added drop-wise. The resulting mixture isstirred at 40° C. overnight. The conjugate is purified by size exclusionchromatography (Sephadex G-25) to yield compound 8.

Example 19 Synthesis of a Chimeric Compound with a Branched Structure

C-94, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages and the spacermoiety is glycerol, connected to the nucleic acid moieties viaphosphorothioate linkages.

C-94: (5′-TCGTCGA-3′-HEG)₂-glycerol-HEG-5′-TCGTCGA-3′

The C-94 molecule was synthesized by TriLink BioTechnologies (SanDiego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturers protocol for 1 umol phosphorothioateDNA. The nucleoside monomers and the spacer moiety precursors[1,3-di-(4,4′-O-dimethoxytrityl)-glycerol-2—O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (symmetrical branched phosphoramiditeobtained from ChemGenes, Ashland, Mass., FIGS. 2) and4,4′-O-dimethoxytrityl-hexaethylene glycol-O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (obtained from Glen Research, Sterling,Va.)] were dissolved in anhydrous acetonitrile to a final concentrationof 0.05 M. The glycerol and HEG spacers were placed in auxiliary monomersites on the instrument. The instrument was programmed to add thenucleotide monomers, HEG spacers, and the glycerol spacer in the desiredorder, with synthesis of the nucleic acid moieties occurring in the 3′to 5′ direction.

-   -   1. Use a 3′-support bound “A” solid support    -   2. Synthesis of 5′-TCGTCGA-3′-moiety    -   3. Addition of HEG spacer    -   4. Addition of the symmetrical branched phosphoramidite based on        glycerol    -   5. Addition of two HEG spacers simultaneously    -   6. Synthesis of two 5′-TCGTCGA-3′ moieties simultaneously

The preparation of this branched compound followed the same protocoldescribed in Example 2, except that in steps 5 and 6, each reagentdelivery in the synthesis cycle was doubled because two nucleic acidchains were built simultaneously. The symmetrical branchedphosphoramidite shown in FIG. 2 requires the nucleic acid sequencessynthesized after the addition of the symmetrical branchedphosphoramidite to be the same, although the nucleic acid sequencesynthesized before its addition may be the same or different from thelater sequences.

The branched compound was purified by HPLC as described in Example 12and characterized as described in Example 2.

C-96 and C-101 were synthesized analogously.

C-103 and C-104 were also synthesized by the same method, except thateither triethylene glycol or propyl spacers were used, respectively, inplace of the hexaethylene glycol spacers.

Example 20 Synthesis of a Chimeric Compound with a Branched Structure

C-98, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with phosphorothioate linkages and the spacermoiety is glycerol, connected to the nucleic acid moieties viaphosphorothioate linkages.

C-98: (5′-TCGTCGA-3′-HEG)₃-trebler-HEG-5′-AACGTTC- 3′-HEG-5′-TCGA-3′

The C-98 molecule was synthesized by TriLink BioTechnologies (SanDiego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturers protocol for 1 umol phosphorothioateDNA. The nucleoside monomers and the spacer moieties [treblerphosphoramidite (obtained from Glen Research, Sterling, Va.) and4,4′-O-dimethoxytrityl-hexaethylene glycol-O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (obtained from Glen Research, Sterling,Va.)] were dissolved in anhydrous acetonitrile to a final concentrationof 0.05 M. The trebler and HEG spacers were placed in auxiliary monomersites on the instrument. The instrument was programmed to add thenucleotide monomers, HEG spacer and the trebler spacer in the desiredorder, with synthesis of the nucleic acid moieties occurring in the 3′to 5′ direction.

1. Use a 3′-support bound “A” solid support

2. Synthesis of 5′-TCGA-3′-moiety

3. Addition of HEG spacer

4. Synthesis of the 5′-AACGTTC-3′ moiety

5. Addition of HEG spacer

6. Addition of the trebler phosphoramidite (see FIG. 2)

7. Addition of three HEG spacers simultaneously

8. Synthesis of three 5′-TCGTCGA-3′ moieties simultaneously

The preparation of this branched compound followed the same protocoldescribed in Example 2, except that in steps 7 and 8, each reagentdelivery in the synthesis cycle was tripled because 3 nucleic acidchains were built simultaneously. The symmetrical treblerphosphoramiditeshown in FIG. 2 requires the nucleic acid sequences synthesized afterthe addition of the symmetrical treblerphosphoramidite to be the same,although the nucleic acid sequence synthesized before its addition maybe the same or different from the later sequences.

The branched compound was purified by HPLC as described in Example 12,and characterized as described in Example 2.

Example 21 Synthesis of a Linear Chimeric Compound with HexaethyleneGlycol Spacers and a 3′-Thiol Linker

CICs containing 3′-thiol linkers are first synthesized and purified astheir disulfide derivatives. The disulfide group is then reduced toyield the reactive thiol group. For example, to synthesize C-116, C-8was synthesized as in Example 2, except that 3′-Thiol Modifier C3 S—SCPG (Glen Research, Sterling, Va.) was used as the solid support insteadof the “T” solid support.

C-116: 5′-TCGTCGA-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′-(CH₂)₃SS(CH₂)₃OH

It will be appreciated that C-116 can be described as[C-8]-3′-disulfide. The CIC was purified by HPLC as described in Example12. The compound was characterized as described in Example 2.

C-116 was reduced to the thiol using tris(2-carboxyethylphosphine)hydrochloride (TCEP; Pierce, Rockford, Ill.). C-116 was dissolved to aconcentration of 30.5 mg/ml (0.8 ml, 24.4 mg; 3.14 umol) in 100 mMsodium phosphate/150 mM sodium chloride/1 mM EDTA/pH 7.4 buffer. In aseparate vial, TCEP was dissolved to a concentration of 0.167 M in 100mM sodium phosphate/150 mM sodium chloride/1 mM EDTA/pH 7.4 buffer. 5equivalents (100 ul, 4.8 mg, 17 umol) of the TCEP stock solution wereadded to the CIC solution. The solution was mixed gently, incubated for120 min at 40° C., and purified on a Sephadex G-25 column (5 ml,Amersham Pharmacia, Piscataway, N.J.) to yield C-117 (13.2 mg). It willbe appreciated that C-117 can be described as [C-8]-3′-thio. The CIC waspurified by HPLC as described in Example 12.

C-115 was synthesized analogously from C-114.

Example 22 Synthesis of a Linear Chimeric Compound with Propyl Spacersand a 5′-Thiol Linker

CICs containing 5′-thiol linkers are first synthesized and purified astheir disulfide derivatives. The disulfide group is then reduced toyield the reactive thiol group. Compound C-110 (below) can be describedas 5′-disulfide-C-11. Compound C-111 can be described as 5′-thiol-C-11.

C-110: HO(CH₂)₆SS(CH₂)₆-5′-TCGTCG-3′-C3-5′- ACGTTCG-3′-C3-5′-AGATGAT-3′

C-110 was synthesized as described in Example 3, except that the finalcoupling was with the thiol modifier C6 S—S (Glen Research, Sterling,Va.). The CIC was purified by HPLC as described in Example 12. Thecompound was characterized as described in Example 2. C-110 was reducedto the thiol using tris(2-carboxyethylphosphine) hydrochloride (TCEP;Pierce, Rockford, Ill.) as described in Example 22.

C-107, C-113 and P-16 were synthesized analogously.

Example 23 Synthesis of a Chimeric Compound with a Branched Structure bya Conjugation Strategy

C-105 was synthesized as shown in FIG. 4. Tris(2-maleimidoethyl)amine(TMEA, Pierce, Rockford, Ill.) was dissolved to a concentration of 4.3mg/ml in dimethylformamide (DMF). The TMEA solution (12 ul, 52 ug, 1.0eq) was added to a solution of C-117 (237 ul, 4.0 mg, 4.0 eq) in 100 mMsodium phosphate/150 mM sodium chloride/1 mM EDTA/pH 7.4 buffer andmixed well. The solution was left at room temperature overnight and waspurified on a Superdex 200 column (24 ml, Amersham Pharmacia,Piscataway, N.J.) in 10 mM sodium phosphate/141 mM sodium chloride/pH7.0 buffer. The product was dried in vacuo, dissolved in 0.4 ml of MilliQ water, and precipitated with 1.0 ml of 95% ethanol. After freezing at−20° C. for 1 hour, the mixture was centrifuged (2 min at 14 K RPM), andthe supernatant was carefully removed. The pellet was dissolved in 0.35ml of Milli Q water and the concentration of C-105 was measured (0.4 mgisolated). The compound was analyzed as described in Example 2.

C-99 was synthesized analogously.

Example 24 Synthesis of a Chimeric Compound with a Branched Structure bya Conjugation Strategy

A. Synthesis of Maleimido-STARBURST DENDRIMER® Generation 2

The STARBURST Dendrimer®, Generation 2, containing 16 hydroxyl groups,was purchased as a 20% solution in methanol from Aldrich (Milwaukee,Wis.). The dendrimer (191 ul, 38.2 mg, 11.7 umol) was dried in vacuo,re-dissolved in 200 ul of DMF and re-dried in vacuo to remove the lasttraces of methanol. To prepare the maleimido-dendrimer,N-(p-maleimidophenyl)isocyanate (PMPI, 50 mg, 233.5 umol) was dissolvedin 200 ul of DMF in a separate glass vial and then quickly added to thedendrimer. The mixture was vortexed until the dendrimer dissolved. Thesolution was put on a rotating mixer overnight at room temperature. Thesolution was concentrated in vacuo, dissolved in 20%methanol/dichloromethane (1 ml), and purified on a 7.5 g silica gelcolumn (70-230 mesh, 60 A) in 20% methanol/dichloromethane. Themaleimido-dendrimer product eluted from the column in the first fraction(due to the presence of residual DMF) and was free of the PMPIby-products. The product was concentrated to a tan solid (10 mg, 13%yield).

B. Synthesis of STARBURST DENDRIMER®-(5′-TGACTGTGAACGTTCGAGATGA)_(x=3-16) (SEQ ID NO:2) (C-102)

The maleimido-dendrimer (5.7 mg) was dissolved in dimethylsulfoxide(DMSO) to form a stock solution at a concentration of 2.5 mg/ml. Themaleimido-dendrimer stock solution (100 ul, 0.25 mg, 0.0375 umol) wasadded to a solution of C-107 (9.1 mg, 1.2 umol) in 100 mM sodiumphosphate/150 mM sodium chloride/1 mM EDTA/pH 7.4 buffer (0.7 ml). Thesolution was placed on a rotating mixer overnight at room temperatureand the product was purified on a Superdex 200 column (24 ml, AmershamPharmacia, Piscataway, N.J.) in 10 mM sodium phosphate/141 mM sodiumchloride/pH 7.0 buffer. The product eluted in the void volume at 10.4min (1.3 mg). The product was found to be a mixture of high molecularweight species, representing different loadings of polynucleotide on thedendrimer, by analysis on a 1.2% agarose E-gel (Invitrogen, Carlsbad,Calif.). C-102 ran as a mixture of products between 1 kb to greater than15 kb (effective size compared to double-stranded DNA markers).

Example 25 Synthesis of a Linear Chimeric Compound with Propyl Spacersand Mixed Phosphodiester/Phosphorothioate Linkages

C-84, having the structure shown below, was synthesized. The nucleicacid moieties are DNA with either phosphorothioate linkages, indicatedby a lower case “s”, or phosphodiester linkages (all other linkages),and the spacer moieties are propyl (C3), connected to the nucleic acidmoieties via phosphodiester linkages.

C-84: 5′-GsGs-3′-C3-5′-TGC-3′-C3-5′-ATCGAT-3′-C3-5′-GCA-3′-C3-5′-GGsGsGsGsG-3′(where a lower case “s” indicates a phosphorothioate linkage and theother linkages are phosphodiester)

The C-84 molecule was synthesized by TriLink BioTechnologies (San Diego,Calif.) on a Perseptive Biosystems Expedite 8909 automated DNAsynthesizer using the manufacturer's protocol for 1 umolphosphorothioate DNA. The phosphorothioate linkages were programmedusing upper case letters for the bases and the phosphodiester linkageswere programmed using lower case letters for the bases and auxiliarypositions containing the propyl spacer phosphoramidite. The nucleosidemonomers and the spacer moiety precursor,4,4′-O-dimethoxytrityl-propyl-O—(N,N-diisopropyl)2-cyanoethylphosphoramidite (obtained from Glen Research, Sterling, Va.)were dissolved in anhydrous acetonitrile to a final concentration of0.05 M. The C3 spacer was placed in an auxiliary monomer site on theinstrument. The instrument was programmed to add the nucleotide monomersand C3 spacers in the desired order, with synthesis of the nucleic acidmoieties occurring in the 3′ to 5′ direction.

1. Use a 3′-support bound “G” solid support

2. Synthesis of 5′-GGsGsGsGsG-3′

3. Addition of C3 spacer

4. Synthesis of 5′-GCA-3′

5. Addition of C3 spacer

6. Synthesis of 5′-ATCGAT-3′

7. Addition of C3 spacer

8. Synthesis of 5′-TGC-3′

9. Addition of C3 spacer

10. Synthesis of 5′-GsGs-3′

The synthesis, deprotection, workup, and analysis were performed asdescribed in Example 2.

C-85 and C-87 were synthesized analogously.

Example 26 Synthesis of Oligonucleotides Containing Fewer Than Eight (8)Nucleotides

Polynucleotides containing fewer than eight bases and containingphosphorothioate linkages were synthesized on a Perseptive BiosystemsExpedite 8909 automated DNA synthesizer. Polynucleotides were purifiedby RP-HPLC on a Polymer Labs PLRP-S column using an increasing gradientof acetonitrile in 0.1 M triethylammonium acetate. The purifiedpolynucleotides were concentrated to dryness, the 4,4′-dimethoxytritylgroup was removed with 80% aqueous acetic acid, and then the compoundswere precipitated two times from 0.6 M aqueous sodium acetate/pH 5.0with 3 volumes of isopropanol. The polynucleotides were dissolved inMilli Q water and the yield was determined from the absorbance at 260nm. Finally, the polynucleotides were lyophilized to a powder. Thepolynucleotides were characterized, and the endotoxin contentdetermined, as described in Example 2.

Example 27 Preparation of Cationic Biodegradable Microcarriers

Cationic poly(lactic acid, glycolic acid) microcarriers (cPLGA) wereprepared as follows. 0.875 g of poly (D,L-lactide-co-glycolide) 50:50polymer (Boehringer Mannheim, Indianapolis, Ind.) with an intrinsicviscosity of 0.41 dl/g (0.1%, chloroform, 25° C.) was dissolved in 7.875g of methylene chloride at 10% w/w concentration, along with 0.3 g ofDOTAP. The clear organic phase was then emulsified into 500 ml of PVAaqueous solution (0.35% w/v) by homogenization at 4000 rpm for 30minutes at room temperature using a laboratory mixer (Silverson L4R,Silverson Instruments). System temperature was then raised to 40° C. bycirculating hot water through the jacket of the mixing vessel.Simultaneously, the stirring rate was reduced to 1500 rpm, and theseconditions were maintained for 2 hours to extract and evaporatemethylene chloride. The microsphere suspension was allowed to cool downto room temperature with the help of circulating cold water.

Microcarriers were separated by centrifugation at 8000 rpm for 10minutes at room temperature (Beckman Instruments) and resuspended indeionized water by gentle bath sonication. The centrifugal wash wasrepeated two additional times to remove excess PVA from the particlesurface. Final centrifugal pellets of particles were suspended inapproximately 10 ml of water, and lyophilized overnight. The driedcationic microcarrier powder was characterized for size and surfacecharge: mean size (number weighted, μ)=1.4; zeta potential (mV)=32.4.

Example 28 Immunomodulation of Human Cells by CICs

Tests were conducted to assess the immunomodulatory activity of (1)chimeric molecules containing spacer moieties and (2) polynucleotides.

The chimeric compounds and polynucleotides were synthesized as describedsupra or by conventional phosphorothioate chemistry. Polynucleotides P-6and P-7 were synthesized by Hybridon Specialty Products (Milford Mass.).Immuno-modulatory activity was determined by routine assays as disclosedherein.

Peripheral blood was collected from volunteers by venipuncture usingheparinized syringes. Blood was layered onto a FICOLL® (AmershamPharmacia Biotech) cushion and centrifuged. PBMCs, located at theFICOLL® interface, were collected, then washed twice with cold phosphatebuffered saline (PBS). The cells were resuspended and cultured in 48well plates (Examples 29-32) or 96-well plates (Examples 33-40) at 2×10⁶cells/mL at 37° C. in RPMI 1640 with 10% heat-inactivated human AB serumplus 50 units/mL penicillin, 50 μg/mL streptomycin, 300 μg/mL glutamine,1 mM sodium pyruvate, and 1×MEM non-essential amino acids (NEAA).

The cells were cultured in the absence of test samples, in the presenceof test samples at 20 μg/ml (0.5 OD/ml), or in the presence of testsamples at 20 μg/ml premixed with 100 μg/ml cPLGA (when used) for 24hours. Cell-free medium was then collected from each well and assayedfor IFN-γ and IFN-α concentrations. SAC (Pansorbin CalBiochem, 1/5000dilution) was used as a positive control. SAC contains is Staph. aureus(cowan) cell material.

IFN-γ and IFN-α were assayed using CYTOSCREENT™ ELISA kits fromBioSource International, Inc., according to the manufacturer'sinstructions.

In the human PBMC assay, background levels of IFN-γ can vary, evensignificantly, with the donor. Levels of IFN-α generally exhibit lowbackground levels under unstimulated conditions.

Examples of results from such assays are shown in Examples 29-40 below.

In each of the experiments shown, “medium alone” and “P-7” are negativecontrols. “P-7” has been previously shown not to have immunostimulatoryactivity. SAC and “P-6” are positive controls. P-6 has been previouslyshown to have significant immunostimulatory activity.

Example 29 Immunostimulatory Activity of CICs

This example shows that four different CICs had significantimmunomodulatory activity as evidenced by stimulation of IFN-γ and IFN-αsecretion (Table 3). As expected, P-7 had no activity. In addition, P-1,a TCG-containing 7-mer, had no activity. Interestingly, CICs with HEGand propyl spacer moieties showed different degrees of stimulation ofIFN-α secretion. Although both types of CICs stimulated IFN-α secretion,the effect was more marked for the HEG-containing CICs.

TABLE 3 IFN-γ (pg/ml) IFN-α (pg/ml) Test compound Donor 1 Donor 2 meanDonor 1 Donor 2 mean medium alone 8 0 4 0 0 0 P-7 410 51 231 0 0 0 SAC2040 1136 1588 393 43 218 P6 2180 669 1425 401 39 220 P-1 8 0 4 0 0 0C-8 1916 696 1306 1609 44 827 C4 2157 171 1164 142 0 71 C-10 1595 9521273 1662 50 856 C-11 2308 270 1289 119 0 59

Example 30 Activity of Polynucleotides

This example shows that polynucleotides P-1, P-2, P-3, P-4 and P-5 didnot have immunomodulatory activity (Table 4). These polynucleotides havethe sequences of the nucleic acid moieties of C-10 and C-11, shown inExample 29 to have immunomodulatory activity.

TABLE 4 IFN-γ (pg/ml) IFN-α (pg/ml) Test compound Donor 3 Donor 4 meanDonor 3 Donor 4 mean medium alone 0 3 2 0 18 9 P-7 3 8 5 0 31 15 SAC1179 2000 1589 50 969 510 P-6 99 223 161 28 106 67 P-1 1 4 2 0 32 16 P-31 3 2 0 32 16 P-4 0 3 1 0 58 29 P-5 0 3 2 0 57 29 P-2 0 4 2 0 40 20

Example 31 Activity of Polynucleotide Mixtures

This example shows a mixture of polynucleotides P-1 and P-3, or P-1,P-3, P-4 and P-5 did not have immunomodulatory activity (Table 5). Thesepolynucleotides have the sequences of the nucleic acid moieties of C-10and C-11 which did have immunomodulatory activity. The mixturescontained equal amounts of each polynucleotide, at a total concentrationof 20 μg/ml total polynucleotide.

TABLE 5 IFN-γ (pg/ml) IFN-α (pg/ml) Test compound Donor 5 Donor 6 meanDonor 5 Donor 6 mean medium alone 3 52 28 20 20 20 P-7 7 66 37 20 94 57SAC 903 284 593 458 8215 4337 P-6 73 1170 621 54 482 268 (P-1) + (P-3) 336 19 20 40 30 (P-1) + (P-3) + 1 99 50 70 65 68 (P-4) + (P-5) C-10 102806 454 91 1700 896 C-11 25 792 409 76 175 126

Example 32 Immunomodulatory Activity of CICs

This example shows the immunomodulatory activity of C-10 and C-11, in anassay with different donors than Examples 29 and 31 (Table 6).

TABLE 6 IFN-γ (pg/ml) IFN-α (pg/ml) Test compound Donor 7 Donor 8 meanDonor 7 Donor 8 mean medium alone 1 0 1 0 0 0 P-7 2 2 2 0 0 0 SAC 5941100 847 22 303 163 P-6 15 367 191 4 59 32 C-10 23 198 111 46 539 293C-11 5 419 212 6 56 31

Example 33 Immunomodulatory Activity of CICs

This example shows immunomodulatory activity of C-8 and C-9, in an assaywith different donors than Example 29 (Table 7). P-2, a TCG-containing6-mer, had no activity.

TABLE 7 IFN-γ IFN-α Donor Donor Donor Donor Donor Donor Donor 9 10 11 12mean Donor 9 10 11 12 mean medium 17 1 1 10 7 4 2 2 15 6 alone P-7 5 2 32 3 0 3 1 5 2 SAC 380 688 159 73 325 2246 364 1129 1029 1192 P-6 66 2072 23 45 12 28 12 12 16 P-2 2 3 1 2 2 0 2 1 4 2 C-8 312 35 31 28 102 5830 18 49 39 C-9 134 7 56 30 56 8 10 1 15 8

Example 34 Immunomodulatory Activity of CICs

The assays shown in Table 8 demonstrate immunostimulatory activity ofseveral CICs of the invention, i.e., CICs characterized by a variety ofdifferent short nucleic acid moieties and a variety of different spacermoieties. Table 8 also shows that compound M-1, which has a mixedHEG/nucleic acid structure but lacks any 5′-C,G-3′ sequence (see Table2), as well as certain other compounds (C-19), did not show activity.The formulation of the CICs with cPLGA significantly enhanced inductionof IFN-α. IFN-γ levels were also increased in some cases. The numbers“28---” represent individual donors.

TABLE 8 Conc IFN-γ (pg/ml) IFN-α (pg/ml) stim ug/ml 28065 28066 2806728068 mean 28065 28066 28067 28068 mean cells alone  0 96 2 1 2 25 0 4 06 3 P-6 20 439 12 28 906 346 14 17 45 126 50 P-7 20 397 1 8 15 105 0 8 03 3 P-2 20 79 1 1 0 20 0 3 0 0 1 P-3 20 94 27 1 0 31 0 0 5 0 1 P-4 20 931 1 1 24 0 0 9 0 2 P-2 + P-3 + P-4 20 tot; 6.7 ea 99 0 1 0 25 0 0 8 0 2C-8 20 1000 19 56 419 373 123 6 96 358 146 C-9 20 1000 8 57 510 394 13 022 64 25 C-10 20 1000 9 51 559 405 116 6 107 340 142 C-17 20 1000 6 32459 374 21 0 22 95 34 C-18 20 1000 102 27 695 456 51 9 16 162 59 C-19 2084 8 1 2 24 0 1 0 13 4 C-20 20 354 13 16 505 222 21 5 13 64 26 C-21 20653 16 24 960 413 227 24 183 769 300 C-23 20 438 5 6 238 172 52 3 19 13753 C-24 20 337 2 4 116 115 28 0 8 67 26 C-25 20 541 6 19 337 226 11 0 2279 28 M-1 20 157 1 40 2 50 0 0 3 0 1 C-27 20 475 3 24 226 182 3 0 24 1611 C-28 20 1082 5 42 410 385 3 0 29 52 21 PLGA  0 55 1 1 5 16 0 2 12 106 P-6 + PLGA 20 975 191 287 573 506 388 194 565 2000 787 P-7 + PLGA 2019 27 6 11 15 0 5 0 0 1 P-2 + PLGA 20 357 138 104 443 261 982 708 21002336 1532 P-3 + PLGA 20 134 1 1 4 35 307 0 0 0 77 P-4 + PLGA 20 19 1 0 36 34 5 0 0 10 P-2 + P-3 + P-4 + 20 tot; 6.7 ea 122 4 14 70 53 1820 0 435106 590 PLGA C-8 + PLGA 20 527 280 245 357 352 2395 538 4380 4625 2985C-9 + PLGA 20 334 139 343 456 318 1093 130 1686 2045 1239 C-10 + PLGA 20619 152 557 420 437 2049 369 3515 3586 2380 C-17 + PLGA 20 508 184 587355 408 1914 240 2729 2774 1914 C-18 + PLGA 20 732 108 355 448 411 2188375 3513 7141 3304 C-19 + PLGA 20 1000 780 730 466 744 5997 3753 143597079 7797 C-20 + PLGA 20 1055 256 270 488 517 1044 191 1265 2000 1125C-21 + PLGA 20 682 874 390 481 607 2468 784 3372 4962 2897 C-23 + PLGA20 216 161 120 377 219 789 189 1573 2000 1138 C-24 + PLGA 20 236 47 188707 295 31 20 772 340 291 C-25 + PLGA 20 427 179 289 499 348 414 87 10821335 730 M-1 + PLGA 20 7 1 3 5 4 0 0 8 5 3 C-27 + PLGA 20 888 205 235466 448 136 44 388 259 207 C-28 + PLGA 20 860 88 489 415 463 216 73 401520 303 SAC  0 1000 339 511 355 551 284 156 1544 350 583

It will be apparent from review of Table 8 that Donor 28065 exhibitedhigh background in the IFN-gamma assay. Values rendered as “1000”indicate a measurement outside the limits of sensitivity of the assay.

Example 35 Activity of CIC Containing 3-Nucleotide Nucleic Acid Moietiesand Enhancement of Activity by cPLGA

This example shows immunostimulatory activity of several CICs in thepresence and absence of cPLGA as assayed using human PBMCs.Interestingly, the phosphodiester version of C-30 (C-31) was inactive asa CIC alone, but had good activity when formulated with cPLGA. In fact,the general trend was that while the CICs containing all phosphodiesterlinkages (C-31, C-36, and C-93) were inactive as CICs alone, they becamesignificantly more active when formulated with cPLGA.

C-32, a CIC containing only trimeric nucleic acid moieties, had activitywhen used alone and demonstrated more activity when formulated withcPLGA. See Table 9.

TABLE 9 IFN-γ (pg/ml) IFN-α (pg/ml) stim 28089 28090 28098 28099 mean28089 28090 28098 28099 mean cells alone 0 0 0 4 1 25 79 33 28 41 P-6 84255 745 125 302 0 62 105 105 68 P-7 0 4 0 2 1 0 27 19 37 21 C-10 35 44174 140 98 17 61 187 304 142 C-21 61 68 218 124 118 56 157 286 466 241C-22 31 15 110 91 62 0 46 97 247 97 C-8 62 52 205 116 109 21 124 314 362205 C-9 12 7 10 10 67 39 C-29 63 50 150 177 110 75 92 359 332 214 C-30 06 12 20 9 134 29 52 47 65 C-31 0 0 0 2 0 158 26 62 29 69 C-32 0 5 11 3513 285 31 46 59 106 C-33 0 0 0 3 1 56 22 34 30 36 C-93 0 0 0 3 1 0 30 2529 21 C-28 14 15 183 45 64 0 64 42 67 43 PLGA 15 2 16 10 11 8 38 39 4933 P-6 + PLGA 606 144 3277 160 1047 197 103 340 91 183 P-7 + PLGA 121 391 5 55 7 85 36 47 44 C-10 + PLGA 804 373 1501 301 745 523 256 509 1317651 C-21 + PLGA 1138 454 1612 630 958 1347 1020 1001 2302 1418 C-22 +PLGA 772 244 1271 357 661 619 386 604 1339 737 C-8 + PLGA 668 332 1863506 842 1005 683 934 1680 1075 C-9 + PLGA 1036 330 683 308 363 335C-29 + PLGA 825 477 1536 341 795 909 711 855 1419 973 C-30 + PLGA 97 233447 41 205 44 116 49 33 60 C-31 + PLGA 256 327 1597 406 647 696 912 10281361 999 C-32 + PLGA 454 192 259 57 240 281 289 218 131 230 C-33 + PLGA171 186 249 96 176 658 1220 1764 1304 1237 C-93 + PLGA 427 628 1707 323771 990 1738 2681 4000 2352 C-28 + PLGA 683 306 2252 224 866 136 155 14170 126 SAC 195 489 101 306 273 67 239 92 70 117

Example 36 Immunostimulatory Activity of CICs Containing 5′ TCG

This example shows immunomodulation by CICs containing various nucleicacid moieties (see Table 10). In general, sequences containing a5′-TCG-3′ (C-8, C-21, C-50, C-51, etc.) or 5′-NTCG (C-46), where N isany nucleoside, were more active than other CG-containing CICs (C-24,C-52). Additionally, while most of the CICs induced a significant amountof IFN-γ, the results were more variable for the induction of IFN-α,suggesting the motif requirements for IFN-α induction may be morestringent than those for IFN-γ induction. In particular, CICs containinga 5′-TCGA-3′ (C-50, C-51, C-45) generated more IFN-α than CICscontaining a 5′-TCGT-3′(C-41, C-42, C-52).

With the exception of C-8 and C-21 (including the motif 5′-TCGTCGA-3′),the best IFN-α induction was generated by CICs with the TCGA in the 5′position.

CICs containing only hexameric (C-22), pentameric (C-43), and tetrameric(C-44) nucleic acid moieties were found to induce IFN-γ when used alone.In addition, each of these CICs, as well as C-32 containing onlytrimeric nucleic acid moieties, induced considerable IFN-γ and IFN-αwhen formulated with cPLGA. C-39, a CIC with two heptameric nucleic acidmoieties, was active when used alone, while C-40, a CIC with onehexameric and one tetrameric nucleic acid moiety, was inactive in thisexperiment. Both of these CICs exhibited significant activity whenformulated with cPLGA.

TABLE 10 IFN-γ (pg/ml) IFN-α (pg/ml) stim 28042 28043 28044 28045 mean28042 28043 28044 28045 mean cells alone 15 4 3 5 7 0 44 9 0 13 P-6 4951189 925 212 705 27 85 36 21 42 P-7 66 76 26 13 45 0 11 22 20 13 C-8 468939 1000 234 660 20 51 32 5 27 C-24 148 156 312 26 161 0 0 8 0 2 C-21790 1519 1198 177 921 57 72 79 15 56 C-42 198 1067 4000 37 1326 0 29 240 13 C-41 174 1075 841 45 534 0 3 23 0 7 C-45 590 1466 984 253 823 62123 152 14 88 C-46 399 814 480 63 439 24 73 26 3 31 C-47 112 537 142 17202 20 0 0 0 5 C-50 1324 1292 509 192 829 36 137 193 35 100 C-51 7951349 1114 411 917 112 245 240 36 158 C-52 238 214 212 28 173 0 3 35 4822 M-1 45 29 7 3 21 0 0 13 2 4 C-22 206 343 736 40 331 12 18 67 30 32C-43 128 536 566 16 312 0 14 20 0 8 C-44 238 359 484 51 283 0 12 60 1 18C-32 91 19 78 17 51 0 0 8 0 2 C-39 343 488 281 137 312 31 187 46 36 75C-40 26 55 20 23 31 0 26 31 2 15 PLGA 192 82 55 3 83 0 0 0 8 2 P-6 +PLGA 1382 1538 2581 178 1420 106 387 371 38 226 P-7 + PLGA 152 324 17412 166 0 2 0 2 1 C-8 + PLGA 1367 2547 1490 286 1423 2182 2193 716 2111325 C-24 + PLGA 1017 1380 1362 52 953 0 31 65 0 24 C-21 + PLGA 40001204 1870 325 1850 2959 2024 886 191 1515 C-42 + PLGA 1515 1417 2190 3721374 425 1081 295 69 468 C-41 + PLGA 710 1940 1910 496 1264 535 1987 534119 794 C-45 + PLGA 1380 2292 1920 634 1557 2408 4000 1693 642 2186C-46 + PLGA 2201 2352 1432 472 1614 502 1309 257 100 542 C-47 + PLGA3579 4000 1137 161 2219 46 271 30 0 87 C-50 + PLGA 2969 1209 1465 4021511 1548 2818 1242 327 1484 C-51 + PLGA 2018 4000 1000 463 1870 18373241 1154 536 1692 C-52 + PLGA 1172 1726 1551 117 1142 12 34 34 0 20M-1 + PLGA 215 159 23 3 100 0 1 0 0 0 C-22 + PLGA 4000 2975 1085 1362049 325 1186 226 42 445 C-43 + PLGA 2210 2594 1354 194 1588 358 1293402 49 526 C-44 + PLGA 1452 4000 2006 276 1934 986 4000 1768 192 1736C-32 + PLGA 2211 4000 2759 133 2276 204 1142 771 12 532 C-39 + PLGA 18004000 2275 274 2087 2167 4000 2613 736 2379 C-40 + PLGA 1438 498 1813 160977 2758 4000 1556 370 2171 SAC 1618 1271 1053 123 1016 285 110 57 0 113

Example 37 Immunostimulatory Activity of CICs

This example shows immunomodulation assays for additional linear CICs(some containing both phosphorothioate (PS) and phosphodiester (PO)linkages) and branched CICs (Tables 11 and 12). Comparison of C-94, abranched CIC, with C-21, a linear CIC containing the same nucleic acidmoieties, showed that the branched CIC induced 4-fold more IFN-α thanthe linear CIC. The amounts of IFN-γ and IFN-α induced weresignificantly increased for each CIC by formulation with cPLGA. Thephosphodiester versions of C-94 and C-93 were active only whenformulated. C-87 showed remarkable induction of IFN-α.

TABLE 11 IFN-γ (pg/ml) IFN-α (pg/ml) stim 28042 28043 28044 28045 mean28042 28043 28044 28045 mean cells alone 11 4 0 13 7 8 2 3 64 19 P-6 3241036 529 653 636 9 34 22 108 43 P-7 34 19 48 35 34 0 0 4 54 15 C-8 623753 646 604 656 78 25 52 256 103 C-53 39 27 38 26 32 0 0 0 5 1 C-49 367433 767 353 480 30 8 100 88 57 C-84 29 23 69 232 88 0 0 5 222 57 C-85 1713 315 134 120 0 0 28 24 13 C-94 443 198 1417 888 736 302 252 664 1855768 C-93 8 1 41 17 17 7 3 81 61 38 C-21 572 460 4000 1644 1669 146 94191 349 195 C-9 691 268 590 1306 714 39 0 11 64 29 PLGA 9 5 59 72 36 7 098 112 54 P-6 + PLGA 601 358 1474 1941 1093 115 116 515 1298 511 P-7 +PLGA 13 13 46 65 34 5 0 0 43 12 C-8 + PLGA 284 551 1781 3113 1432 595396 1013 2259 1066 C-53 + PLGA 21 12 217 210 115 19 0 0 42 15 C-49 +PLGA 1471 1219 4000 2061 2188 904 460 4000 1040 1601 C-84 + PLGA 235 232291 956 428 1777 914 4000 3641 2583 C-85 + PLGA 313 294 554 1167 5822116 921 4000 2413 2362 C-94 + PLGA 2412 755 4000 3379 2637 1883 16404000 4000 2881 C-93 + PLGA 880 316 869 1251 829 778 471 2045 988 1071C-21 + PLGA 4000 690 4000 2533 2806 712 577 2572 1571 1358 C-9 + PLGA1451 763 4000 1804 2005 389 199 397 477 366

TABLE 12 IFN-g (pg/ml) IFN-a (pg/ml) stim 28218 28219 28220 28221 mean28218 28219 28220 28221 mean cells alone 5 5 5 5 5 32 32 32 32 32 P-6 137 25 141 47 32 32 32 32 32 P-7 5 5 5 5 5 32 32 32 32 32 C-87 83 24 38977 281 3075 32 4269 265 1910 C-94 15 39 44 269 92 32 167 633 412 311SAC 2552 621 1383 647 1301 483 105 32 452 268

Example 38 Position of Sequence Motif in CIC

This example describes immunomodulation assays for a number of CICs(some of which were assayed in different donors in previous examples)and illustrates the effect of nucleic acid sequence position in a CIC.

The CICs tested included CICs containing two different CG-containingnucleic acid sequences in nucleic acid moieties (TCGTCGA and ACGTTCG)along with one nucleic acid moiety not containing a CG sequence(AGATGAT). Of the CG-containing nucleic acid sequences, CIC's containinga TCGTCGA sequence have greater activity than CIC's containing onlyACGTTCG. Of these two, CICs with TCGTCGA were more active. The generalstructure of the CICs used in this example, N₁—S₁—N₂—S₂—N₃, can be usedto describe the placement of the motifs within the CIC. Placing the mostactive motif, TCGTCGA, in the N₁ position led to the most active CICs(C-8, C-56). Placement in the N₂ position also conferred activity. Forinstance, C-57 with the TCGTCGA in the N₂ position was somewhat moreactive than C-58, with the TCGTCGA in the N₃ position. A CIC with aACGTTCG sequence in the N₁ position, while being less active than asimilar CIC with a TCGTCGA sequence, was more active than a CIC with thesequence AGATGAT, in the N₁ position (compare C-57 and C-58 to C-59 andC-60). In this experiment, C-61, which contained nucleic acid moietiesthat comprise CG motifs, but not TCG motifs, induced IFN-γ, whenformulated with cPLGA. See Table 13.

TABLE 13 IFN-γ (pg/ml) IFN-α (pg/ml) stim 28156 28157 28158 28159 mean28156 28157 28158 28159 mean cells alone 125 3 4 5 34 3 3 1 8 4 P-6 1132872 207 231 611 52 484 7 32 144 P-8 255 20 31 31 84 16 9 24 8 14 C-81612 742 340 197 723 102 755 61 160 270 C-9 1162 729 192 329 603 26 14278 20 67 C-23 733 576 202 295 452 26 235 59 169 122 C-54 297 378 88 218245 8 96 8 13 31 C-55 511 566 55 186 329 9 5 63 3 20 C-56 1223 543 203563 633 98 415 57 131 175 C-57 419 323 67 262 268 5 52 61 42 40 C-58 404288 59 84 209 13 30 29 23 24 C-60 304 209 26 38 144 5 22 3 1 8 C-61 92179 35 63 92 3 0 47 0 13 PLGA 43 63 5 11 30 85 246 0 3 83 P-6 + PLGA1070 2643 251 496 1115 582 2948 418 359 1077 P-8 + PLGA 95 115 26 34 6743 8 2 23 19 C-8 + PLGA 1083 1862 269 1129 1086 4000 4877 857 1573 2827C-9 + PLGA 814 1412 307 992 881 1398 1778 418 483 1019 C-23 + PLGA 825865 182 1423 824 1020 1621 240 597 869 C-54 + PLGA 838 1150 157 1751 974752 1265 147 278 611 C-55 + PLGA 1048 960 247 2356 1153 505 801 78 211399 C-56 + PLGA 792 604 321 4000 1429 4000 4000 852 2433 2821 C-57 +PLGA 1027 814 101 3056 1250 555 1476 10 252 573 C-58 + PLGA 804 1065 1351021 756 179 932 3 139 313 C-60 + PLGA 650 858 56 1014 645 71 118 32 5068 C-61 + PLGA 1265 1508 238 864 969 4 80 1 63 37 SAC 780 1184 83 659677 208 55 6 34 76

This experiment also compared immunomodulatory activity of two types ofbranched CICs: C-94 has HEG moieties between the branching glycerolcomponent and the nucleic acid moieties, while C-28 has the nucleic acidmoieties attached directly to the glycerol spacer. See Table 14.Interestingly, while the induction of IFN-γ was similar for bothbranched CICs, the induction of IFN-α was dramatically higher for theCIC containing the HEG spacers. A branched CIC, containing three P-6sequences attached via their 5-ends to a maleimido-activatedtriethylamine spacer (C-99), induced IFN-γ only when formulated withcPLGA and did not induce IFN-α. In general, the greatest IFN-αproduction was produced using CICs with nucleic acid moieties attachedvia a branched structure and having multiple unattached or “free”5′-ends of nucleic acid moieties, and including spacers that provideconformational flexibility and distance between the nucleic acidmoieties.

TABLE 14 IFN-γ (pg/ml) IFN-α (pg/ml) stim 110 112 119 120 mean 110 112119 120 mean cells alone 44 24 20 28 29 20 200 2 2 56 P-6 1508 344 144104 525 50 172 234 72 132 P-7 124 24 16 40 51 2 32 474 2 128 C-8 1152540 136 48 469 196 30 264 42 133 C-59 256 52 28 40 94 2 6 2 2 3 C-631536 376 80 60 513 294 38 464 228 256 C-50 1096 264 52 48 365 716 84 838636 569 C-51 1528 240 52 40 465 1408 72 2200 622 1076 C-45 880 192 52 36290 446 130 1074 428 520 C-41 512 100 32 32 169 58 2 182 6 62 C-42 1508204 56 56 456 250 26 156 36 117 C-46 1224 400 68 36 432 58 2 208 48 79C-52 472 48 40 28 147 2 2 292 2 75 C-39 604 116 108 32 215 674 26 444250 349 C-40 180 12 4 20 54 6 198 152 2 90 C-94 5168 284 104 120 14191608 144 2610 878 1310 C-28 5564 52 44 60 1430 38 4 56 26 31 C-99 276 1216 40 86 22 4 86 2 29 PLGA 32 8 72 120 58 10 2 60 92 41 P-6 + PLGA 1640968 960 2300 1467 948 260 1298 1470 994 P-7 + PLGA 72 16 32 316 109 1414 22 2 13 C-8 + PLGA 1948 1220 1188 2384 1685 6674 1138 2130 2650 3148C-59 + PLGA 680 824 620 1828 988 234 2 76 278 148 C-63 + PLGA 1208 15802340 2092 1805 4148 738 2796 2298 2495 C-50 + PLGA 812 3684 1432 9921730 3768 1414 4161 3402 3186 C-51 + PLGA 1240 11216 2896 924 4069 52441260 5104 6148 4439 C-45 + PLGA 2736 3024 3056 2472 2822 5532 1544 54744206 4189 C-41 + PLGA 3168 1808 16000 3656 6158 3542 746 2074 2094 2114C-42 + PLGA 1612 2032 10212 1908 3941 3462 1030 2118 2054 2166 C-46 +PLGA 3048 2012 3720 3608 3097 2372 638 2372 2682 2016 C-52 + PLGA 10321236 2344 1724 1584 64 20 252 206 136 C-39 + PLGA 2024 1332 8228 12443207 3764 846 3078 2658 2587 C-40 + PLGA 1360 1244 5364 1864 2458 2362684 3794 2616 2364 C-94 + PLGA 2668 3188 8840 3396 4523 5658 1838 80006346 5461 C-28 + PLGA 2104 2568 3572 1320 2391 302 2 284 198 197 C-99 +PLGA 768 672 5316 472 1807 114 80 344 260 200

Example 39 Activity of Branched CICs

This example demonstrates that branched CICs with multiple free 5′-endsand conformational flexibility provided by HEG spacers induced moreIFN-α relative to linear CICs with HEG spacers (compare C-94 with C-21and C-96 with C-23) or branched CICs without additional (HEG) spacers(compare C-94 with C-28 and C-96 with C-27). Adding another HEG spacerand a 4-base nucleic acid moiety to C-96 caused a reduction of IFN-αinduction (compare C-96 with C-97). See Table 15.

Immunostimulatory activity of two CICs containing trimeric 5′-TCG-3′motifs was tested (C-91 and C-68). While neither CIC was active on itsown, C-91 had significant activity when formulated on cPLGA.

A hydrophilic polyamide-containing STARBURST Dendrimer® with multipleP-6 sequences conjugated to it (C-102), had significantly more IFN-αactivity than the P-6 sequence alone, when compared with an equal amountof P-6 (on a P-6 strand per strand basis). This result confirms, using adifferent composition and synthetic protocol from that demonstratedabove, the utility of a multimeric delivery of 5′-CG-3′-containingnucleic acid moieties on a flexible, hydrophilic core for significantlyincreased induction of IFN-α.

TABLE 15 IFN-g (pg/ml) IFN-a (pg/ml) stim 28185 28186 28187 28188 mean×4 28185 28186 28187 28188 mean ×2 cells 5 1 13 1 5 20 36 4 32 4 19 38alone P-6 205 17 148 8 94 378 120 41 4 4 42 84 P-7 0 4 19 2 6 25 4 25 531 16 32 C-8 154 25 123 9 78 311 196 31 202 4 108 217 C-94 181 61 384 17161 644 1895 239 136 13 571 1142 C-28 162 24 75 5 66 266 14 4 0 4 6 11C-21 244 37 125 7 103 413 443 64 1 4 128 256 C-23 42 14 29 3 22 88 83 2759 55 56 112 C-27 49 3 21 3 19 75 4 4 39 4 13 25 C-96 163 22 195 12 98392 2550 446 118 40 788 1577 C-97 259 16 125 5 101 405 307 71 4 2 96 192C-9 189 24 95 11 80 319 25 16 4 146 48 95 C-86 1 4 30 5 10 40 7 4 4 3111 22 C-91 3 4 6 7 5 20 9 46 37 4 24 48 C-68 0 4 2 1 2 7 4 13 25 4 11 23C-102 158 43 101 6 77 307 1880 187 109 4 545 1090 PLGA 10 4 13 4 8 30 44 0 4 3 6 P-6 + 315 64 128 39 137 546 710 116 78 4 227 454 PLGA P-7 + 73 15 2 7 27 4 4 4 9 5 10 PLGA C-8 + 319 127 242 24 178 712 1599 646 60135 720 1441 PLGA C-94 + 391 118 280 34 206 823 6761 19553 3949 207 761815235 PLGA C-28 + 395 65 175 13 162 649 84 145 15 13 64 128 PLGA C-21 +333 49 177 20 145 579 3581 3169 1340 64 2038 4077 PLGA C-23 + 199 67 10215 96 382 599 250 110 21 245 490 PLGA C-27 + 292 170 95 14 142 570 54 584 39 39 78 PLGA C-96 + 400 186 244 41 218 872 27504 5572 2464 173 892817857 PLGA C-97 + 356 177 124 39 174 696 2264 668 285 48 816 1632 PLGAC-9 + 384 82 93 19 145 579 479 451 193 35 290 579 PLGA C-86 + 11 3 84 425 101 33 4 4 4 11 22 PLGA C-91 + 161 101 114 1 94 377 880 494 316 4 423847 PLGA C-68 + 31 8 24 4 17 67 14 51 4 4 18 37 PLGA C-102 + 774 132 3807 323 1293 2094 397 221 26 684 1369 PLGA SAC 195 22 274 15 127 506 73 4151 102 82 165

Example 40

This experiment examined the activity of a series of CICs containing ahexameric nucleic acid motif, 5′-TCGTCG-3′, and multiple spacersattached to the 3′-end of the nucleic acid moiety (C-13, C-14, C-15 andC-16). See Table 16. None of the CICs was active when used alone,however all had significant activity when formulated on cPLGA.

TABLE 16 IFN-γ (pg/ml) IFN-α (pg/ml) stim 28057 28058 28059 28060 mean28057 28058 28059 28060 mean cells alone 1 2 1 39 11 0 0 0 0 0 P-6 83103 1230 85 375 621 396 145 123 321 P-7 1 2 3 4 2 0 0 0 0 0 C-13 2 3 125 6 31 0 249 0 70 C-14 3 1 1 3 2 0 0 0 0 0 C-15 5 3 0 1 2 0 0 0 0 0 C-161 2 0 6 2 0 0 0 0 0 PLGA 40 32 49 254 94 35 222 41 0 74 P-6 + PLGA 20002000 2000 452 1613 2000 2000 1747 403 1537 P-7 + PLGA 40 271 16 40 92 0527 161 108 199 C-13 + PLGA 2000 262 221 168 663 5865 7994 5912 14375302 C-14 + PLGA 2000 359 732 2000 1273 3937 6871 6371 2953 5033 C-15 +PLGA 2000 585 2000 258 1211 2991 6282 4138 1731 3786 C-16 + PLGA 172 207277 71 182 1842 2529 2333 1362 2017 SAC 673 2000 2000 2000 1668 920 2000387 146 863

Example 41 Effects of CICs in B-Cell Proliferation Assay

Human PBMCs were isolated from heparinized blood from two normalsubjects. Some PBMCs were held in reserve while the remainder wasincubated with CD19+ MACS beads (Miltenyi Biotec). These cells were thenpassed through a magnet, separating the CD19+ B cells through positiveselection. This population was >98% CD19+ as determined by FACSanalysis. B cells were then cultured at 1×10⁵/200 μl/well in 96-wellround-bottomed plates. In some cases, PBMCs were also cultured, but at2×10⁵/200 μl/well. Cells were stimulated in triplicate with 2 μg/mlpolynucleotide or CIC. The culture period was 48 hours at 37° C. At theend of the culture period, the plates were pulsed with ³H-thymidine, 1μCi/well, and incubated for an additional 8 hours. Then the plates wereharvested using standard liquid scintillation techniques and data wascollected in counts per minutes (cpm).

Experiment A:

The results of Experiment A (Table 17) demonstrate that polynucleotides(P-6) and CICs (C-8, C-9, C-21, C-28) containing 5′-C,G-3′ motifs causeB cells to proliferate. Control compounds, P-7 and M-1, and a heptamericpolynucleotide, P-1, generated little to no B cell proliferation. Thebranched CIC, C-28, and the CIC containing the propyl spacers, C-9,induced more B cell proliferation than CICs containing hexaethyleneglycol spacers, C-8 and C-21. The proliferation of PBMCs mirrored thatof B cells.

TABLE 17 Donor 146 Donor 147 mean cell type stim cpm1 cpm2 cpm3 meancpm1 cpm2 cpm3 mean of both B cells cells alone 538 481 795 605 482 360296 379 492 B cells P-6 29280 33430 30056 30922 35729 18032 21166 2497627949 B cells P-7 4858 5810 7079 5916 4364 4066 2774 3735 4825 B cellsP-1 761 608 721 697 569 460 687 572 634 B cells C-8 23815 30066 2296925617 20914 22370 23659 22314 23966 B cells C-9 35365 42705 45231 4110055543 49035 44985 49854 45477 B cells C-21 28467 16074 19258 21266 1760418851 19887 18781 20024 B cells M-1 1514 2815 1173 1834 1679 1667 14361594 1714 B cells C-28 50999 54630 46418 50682 65593 51040 50357 5566353173 PBMCs cells alone 2744 2303 2284 2444 1301 2402 2143 1949 2196PBMCs P-6 22067 23740 28099 24635 26436 23830 17531 22599 23617 PBMCsP-7 7620 8362 9686 8556 9783 9841 10476 10033 9295 PBMCs P-1 9724 30412425 5063 1706 1960 324 1330 3197 PBMCs C-8 47202 40790 44811 4426838845 39733 27981 35520 39894 PBMCs C-9 55348 24857 39953 40053 8810665413 90665 81395 60724 PBMCs C-21 30338 22685 22383 25135 28819 53037088 22146 23641 PBMCs M-1 8753 5203 4496 6151 1034 3298 1674 2002 4076PBMCs C-28 94977 121595 84977 100516 103916 91439 100905 98753 99635

Experiment B:

Experiment B (Table 18) evaluated the effects of the spacer composition,as well as the CIC structure (linear vs. branched), on B cellproliferation. Linear CICs containing propyl, butyl, abasic, andhydroxymethylethyl spacers tended to induce more B cell proliferationthan the corresponding CICs containing either hexaethylene glycol ortriethylene glycol spacers (compare C-10, C-11, C-17, C-18, C-20, C-25).The dodecyl spacer rendered the CIC inactive (C-19). Notably, the B cellproliferation data does not necessarily mirror the cytokine data shownabove, with particular differences see between B cell proliferation andIFN-α induction.

TABLE 18 PROLIFERATION ASSAY 121 194 mean sample cell stim cpm1 cpm2cpm3 mean cpm1 cpm2 cpm3 mean of both 1 B cells cells alone 451 757 297502 203 228 151 194 348 2 B cells P-6 19996 15031 19804 18277 1367812732 9003 11804 15041 3 B cells P-7 1623 1821 2901 2115 1992 1593 16861757 1936 4 B cells C-8 2604 12078 17696 10793 9333 9391 7602 8775 97845 B cells C-9 21938 35400 23877 27072 13660 16717 17866 16081 21576 6 Bcells C-10 15142 14136 16158 15145 7480 5458 5943 6294 10720 7 B cellsC-11 30367 30412 18528 26436 16967 20898 11253 16373 21404 8 B cellsC-22 17147 14014 6844 12668 6472 5540 3894 5302 8985 9 B cells C-9411418 14406 11110 12311 7361 8505 5349 7072 9692 10 B cells C-28 3539326954 26780 29709 21588 13691 15691 16990 23350 11 B cells C-17 2797530426 9895 22765 17467 14890 10518 14292 18529 12 B cells C-18 1708514653 15869 10028 12217 10538 10928 13398 13 B cells C-19 858 1099 926961 371 403 312 362 662 14 B cells C-20 31276 30851 28532 30220 1808218705 17481 18089 24155 15 B cells C-23 10628 16221 20087 15645 87306532 9596 8286 11966 16 B cells C-24 8206 6789 2799 5931 3979 3407 34683618 4775 17 B cells C-25 34360 35016 26480 31952 16060 19509 1738417651 24802

Example 42 Immunomodulation of Mouse Cells by CIC

Polynucleotides and chimeric compounds were tested for immunostimulatoryactivity on mouse splenocytes. Immunostimulation was assessed bymeasurement of cytokine secretion into the culture media. Cytokinelevels in the culture supernatant were determined by enzyme-linkedimmunosorbent assay (ELISA) tests.

Cells were isolated and prepared using standard techniques. Spleens of 8to 20 week-old BALB/c mice were harvested and the splenocytes isolatedusing standard teasing and treatment with ACK lysing buffer fromBioWhittaker, Inc. Four spleens were pooled in this experiment. Isolatedcells were washed in RPMI 1640 media supplemented with 2%heat-inactivated fetal calf serum (FCS), 50 μM 2-mercaptoethanol, 1%penicillin-streptomycin, and 2 mM L-glutamine and resuspended atapproximately 7×10⁵ cells/ml in 10% FCS/RPMI (RPMI 1640 media with 10%heat-inactivated FCS, 50 μM 2-mercaptoethanol, 1%penicillin-streptomycin, and 2 mM L-glutamine).

Cell cultures were set up in triplicate with approximately 7×10⁵cells/well in a 96-well flat microtiter plate in 100 μl 10% FCS/RPMIwith the cells allowed to rest for at lest 1 hour after plating. Theindicated test compounds were incubated (at the indicatedconcentrations) for 24 hours at 37° C. Cell supernatants were harvestedand frozen at −80° C. Cytokine production by the cells was determined byELISAs, as shown in 19.

TABLE 19 Test Compound Dose IL-6 IL-12 IFNγ P-6 5.0 μg/ml 9311 5374 25051.0 μg/ml 5760 4565 2175 0.1 μg/ml 121 1665 187 C-10 5.0 μg/ml 3342 2329199 1.0 μg/ml 1761 1738 104 0.1 μg/ml 9 122 9 C-11 5.0 μg/ml 10098 42793342 1.0 μg/ml 11814 4914 3220 0.1 μg/ml 458 3359 960 P-7 5.0 μg/ml 9177 23 1.0 μg/ml 7 143 30 SAC 734 1343 18843 media alone 9 124 9

Example 43 Induction of Immune-Associated Genes in the Mouse Lung afterIntranasal Treatment with CICs

The ability of C-9, C-23, and P-6 (positive control) to induce mRNAexpression of 75 different genes in the mouse lung was investigated. Thegenes evaluated included genes encoding cytokines, chemokines, cellsurface molecules, transcription factors, metalloproteases, and othermolecules. The study was performed at Northview Pacific Laboratories(Hercules, Calif.) with 6-8 week old female BALB/c mice from JacksonLabs (Bar Harbor, Me.). Five mice per group were intranasally treatedunder light isoflorane anesthesia with 20 ug of C-9, C-23, P-6 (positivecontrol) or P-7 (negative control) in 50 uL of saline. Previousexperiments demonstrated that optimal induction of most genes was at 6hrs after treatment. Therefore, at 6 hrs the lungs were harvested andsnap-frozen in liquid nitrogen and stored at −80° C. for later use.Total RNA was isolated using RNeasy mini kits (Qiagen Inc., Valencia,Calif.). The RNA samples were DNAse-treated (Roche Diagnostics,Mannheim, Germany) and converted into cDNA using Superscript II RnaseH-Reverse Transcriptase (Invitrogen, Rockville Md.) as described inScheerens et al., 2001, Eur. J. of Immunology 31:1465-74. The cDNAsamples were pooled per group and in each pooled sample the expressionof mRNA of 75 genes was measured using real-time quantitative PCR (ABIPrism 5700, Perkin Elmer Applied Biosystems) and syber green (QiagenInc.). In addition to the genes of interest, in each sample the mRNAexpression of a housekeeping gene was measured (HPRT or ubiquitin). Inorder to correct for the amount of RNA in each sample, all data werecalculated relative to the expression of the housekeeping gene. Aselection of the most upregulated genes is shown in FIG. 5, with dataexpressed as fold-induction over the response in control-treated (P-7)mice. The data demonstrate that C-9, C-23, and P-6 potently induce theexpression of a variety of genes including IL-6, IL-12p40, IFN-alpha,IP-10, and IL-10. Treatment of mice with C-9, however, inducedconsiderably higher mRNA expression of IFN-alpha when compared to theC-23 or P-6 treated group.

Example 44 In Vivo Activity of CICs

An in vivo study was performed by injecting mice (10 mice/group)subcutaneously in the scruff of the neck with 20 ug (200 ul volume) ofP-6 (positive control), P-7 (negative control), C-9, C-23, P-1 or P-11.Blood was collected 2 hours later. For the LPS positive control group,mice were injected intraperitoneally with a 200 ul volume, and blood wascollected 1.5 hours later (i.e., at the peak of LPS induced TNF-αactivity). The blood was clotted and the serum was prepared and storedat −80° C. until assayed. Serum cytokines were assayed using Biosourcecytoscreen kits for TNF-α and Pharmingen antibody pairs for mIL-6 andmIL-12. All samples were assayed in duplicate.

P-6 and the two CICs, C-9 and C-23, each induced IL-12 p40, IL-6, andTNF-α, while the control oligonucleotide, P-7, was inactive (FIG. 6A-C).CIC C-23 was more potent than C-9 and P-6 in this assay. As expected,the hexamer (P-11: 5′-AACGTT) and heptamer (P-1: 5′-TCGTCGA) wereinactive.

Example 45 Primate Immune Response to Antigen and CICs

Immune responses to administration of hepatitis B surface antigen(HBsAg) in the presence of CICs were examined in baboons.

HBsAg was recombinant HBsAg produced in yeast. Groups of baboons (eightanimals per group) included male and female baboons with weights rangingfrom 8-31 kg (group mean weights at 13-16 kg) at the start of the study.

The baboons were immunized two times, at a two-month interval (0 and 2months), by intramuscular injection (IM) with 20 μg HBsAg in a 1 mlvolume. As outlined below, some of the groups also received CICs (C-8 orC-9) or a positive control (P-6) with the HBsAg.

Bleeds on all animals were collected prior to immunization and at 2weeks post-immunization. Anti-HBsAg IgG titers were measured as follows.Baboon serum samples were analyzed by AUSAB EIA commercial kit (AbbottLabs Cat. #9006-24 and 1459-05) using human plasma derived HBsAg coatedbeads. Samples were tested along with a panel of human plasma derivedHBsAg positive and negative standards ranging from 0-150 mIU/ml. Biotinconjugated HBsAg and rabbit anti-biotin-HRP conjugated antibody was usedas the secondary antibody complex used for detection. The assay wasdeveloped with ortho-phenylenediamine (OPD) and the absorbance valueswere determined at 492 nm with background subtraction at 600 nm (QuantumII spectrophotometer, Abbott Labs). Using the specimen absorbance valuethe corresponding concentration of anti-HBsAg is expressed inmilli-international units per ml (mIU/ml) as determined from thestandard curve according to parameters established by the manufacturer.For diluted specimens, quantitation was based on the specimen absorbancethat resulted in a value between 0-150 mIU/ml, multiplying by thedilution factor to arrive at the final concentration.

Statistical analysis was done with log-transformed data by analysis ofvariance (NCSS97 Statistical Software program, Kaysville, Utah) usingOne-Way ANOVA Planned Comparison (α=0.05). p 0.05 was consideredsignificant.

The animal groups tested were immunized as follows:

Group 1-20 μg HBsAg;

Group 2-20 μg HBsAg+1000 μg P-6;

Group 3-20 μg HBsAg+1000 μg C-8;

Group 4-20 μg HBsAg+1000 μg C-9

Results from the study are shown in Table 20 below. Administration ofCICs or the positive control P-6, in conjunction with HBsAg resulted inincreased titers of anti-HBsAg antibodies as compared to administrationof HBsAg alone. In a pairwise comparison, the immune response detectedin Groups 2, 3, and 4 were significantly different from that detected inGroup 1 (p<0.05 for Group 2 and p<0.005 for Groups 3 and 4, post-secondimmunization). There was no statistical difference found between groups2, 3, and 4.

TABLE 20 Baboons Antibody Response (AUSAB EIA) HBsAg + CIC Anti-HBsAgGroup # (mIU/ml) # Vaccine post-first post-second B339 1 0  7 B340 HBV 063 B341 (20 ug) 0 15 B342 0 80 B343 0 55 B344 0 50 B345 0 28 B346 0 24Mean 0 40 Stdev 0 26 B347 2 0 329  B348 HBV 6 121  B349 (20 ug) 0 108 B350 P-6 17 13,569    B351 (1000 ug) 0 315  B352 0 38 B353 15 1,446  B354 21 1,675   Mean 7 2200*  Stdev 9 4,637   B379 3 2 184  B380 HBV 03,038   B381 (20 ug) 0 41,706    B382 C-8 125 3,718   B383 (1000 ug) 0250  B384 52 13,750    B385 0 11,626    B386 0 79 Mean 22 9294** Stdev45 14,121    B387 4 0 5,605   B388 HBV 42 8,978   B389 (20 ug) 0 312 B390 C-9 0 2,992   B391 (1000 ug) 405 12,663    B392 26 112  B393 752,364   B394 0 52 Mean 68 4135** Stdev 139 4,633   *p < 0.05, **p <0.005 compared to HBV alone (Group 1)

Example 46 In Vivo Responses Generated by a CIC—Antigen Conjugate

This example shows the induction of an antibody-mediated immune responsein mice by administration of a CIC-antigen conjugate.

As described below, 10 mice/group were immunized twice intradermally (inthe tail) at two week intervals with C-11/Amb a 1 conjugate synthesizedas described below (1 ug or 10 ug), P-6/Amb a 1 (1 ug) or Amb a 1 (1ug). Anti-Amb a 1-specific IgG1 and IgG2a titers were determined fromsera taken 2 weeks post each injection. In vitro re-stimulations weredone on spleen cells at 6 weeks post 2^(nd) immunization to determineAmb a 1-specific IFNγ and IL-5 responses.

Mice immunized with the C-11-Amb a 1 conjugate showed the characteristicimmune response pattern seen with the P-6-Amb a 1 reference material,specifically, a switch from a Th2, toward a Th1-type Amb a 1-specificimmune response. Mice immunized with either the C-11 or P-6 conjugatesdeveloped strong IgG2a responses and reduced IgG1 responses. Theconjugate treated groups also demonstrated a shut down of the IL-5response and elevation of the IFNγ response. Additionally, the immuneresponses to the C-11-Amb a 1 conjugate appear to increase in a dosedependant fashion, as demonstrated by comparing the 1 ug and 10 ug dosegroups. The C-11-Amb a 1 conjugate elicits an immune response of similarquality to that seen with P-6-Amb a 1.

Results are shown in Tables 21-23.

General Procedure

The animal study was performed at Northview Pacific Laboratories(Hercules, Calif.) using 8-12 week old female BALB/c mice from CharlesRiver Laboratories (Hollister, Calif.). 10 mice/group were injectedtwice intradermally in the tail (ID) at two-week intervals with one ofthe following materials: C-11/Amb a 1 conjugate (1 ug), C-11/Amb a 1conjugate (10 ug), P-6/Amb a 1 conjugate (1 ug) or Amb a 1 antigen (1ug). Bleeds were collected via the retro-orbital route two weeks aftereach of the injections and serum prepared for antibody determination.Six weeks after the 2^(nd) injection spleens were harvested for in vitrore-stimulation assays to determine cytokine response of IFNγ and IL-5.Spleens were assayed individually. Amb a 1 was used at 25 and 5 ug/mlfor re-stimulation with 5×10⁵ cells/well and supernatants harvested onDay 4 and stored at −80° C. until assayed. Controls for the in vitroassay included SAC at 0.01% and PMA/IO at 10 ng/ml and 1 uM,respectively.

Mouse anti-Amb a 1 IgG1 and IgG2a Assays

Mouse serum samples were analyzed by ELISA in 96-well round-bottomplates that were coated with 50 Amb a 1 antigen at 1 μg/ml. Goatanti-mouse IgG1 (or IgG2a) biotin conjugated antibody was used as thesecondary antibody. Streptavidin-horseradish peroxidase conjugate wasused for detection. The assay was developed with TMB and the absorbancevalues were determined at 450 nm with background subtraction at 650 nm(Emax precision microplate reader, Molecular Devices, Sunnyvale,Calif.). The titer was defined as the reciprocal of the serum dilutionthat gave an ELISA absorbance of 0.5 OD using 4-parameter analysis(Softmax Pro97, Molecular Devices, Sunnyvale, Calif.). All samples weretested in duplicate wells on separate plates, and the titers werereported as the mean of the two values.

Mouse IL-5 and IFN-gamma Assays

Supernatants were tested for IL-5 and IFNγ levels by capture ELISA onanti-cytokine monoclonal antibody coated plates. Biotinylatedanti-cytokine MAbs were used as secondary antibodies.Streptavidin-horseradish peroxidase conjugate was used for detection andthe assay was developed with TMB. Concentration was calculated from astandard curve assayed on each plate. The absorbance values weredetermined at 450 nm with background subtraction at 650 nm (Emaxprecision microplate reader, Molecular Devices, Sunnyvale, Calif.). Allsamples were tested in duplicate wells on separate plates, and theconcentrations were reported as the mean of the two values.

Statistics were done on log transformed data with the NCSS97 program(NCSS Statistical Software, Kaysville, Utah) using One-Way ANOVA withPlanned Comparisons, α=0.05. For the following study, p<0.05 isconsidered statistically significant.

Synthesis of the C-11/Amb a 1 Conjugate

Synthesis of Activated C-11 (C-111):

The 5′-disulfide-C-11 (C-110) was dissolved in activation buffer (100 mMsodium phosphate/150 mM sodium chloride/pH 7.5) and activated byreduction with TCEP. The activated CIC (C-111) was purified using a 5 mlSephadex G25 column (Pharmacia) using the same activation buffer asmobile phase. Fractions were collected manually at 0.5-minute intervalsstarting at baseline rise. After purification, the concentration of thevarious fractions was determined using A260 and an extinctioncoefficient of 25.6 OD/mg.

Synthesis of Activated Amb a 1:

Amb a 1 was activated by first blocking the its free-sulfhydryls, andthen adding a hetero-functional cross-linker. Excess reagents wereremoved by desalting using a HiTrap G-25 desalting column (PharmaciaCatalog #17-1408-01). The resulting activated Amb a 1 had an average of9.3 sites per protein activated.

Synthesis of the C-11/Amb a 1 Conjugate:

The activated C-11 (C-111) and activated Amb a 1 were combined and theresulting C-11/Amb a 1 conjugate was fractionated using a Superdex 200size exclusion chromatography column (Pharmacia Cat. #17-1088-01; 1cm×30 cm). Formulation buffer (10 mM phosphate, 150 mM NaCl, pH 7.2) wasused as mobile phase. Fractions were collected at 1-minute intervals,starting when the baseline began to rise.

The conjugate samples were analyzed by SDS-PAGE using a 4-12% NuPAGE gel(Invitrogen, Catalog #NP0322) using MOPS buffer (Invitrogen, Catalog#NP0001), and by Size Exclusion Chromatography (SEC-HPLC) using a BioSepSEC-S3000 column (Phenomenex, Catalog #00H-2146-E0). After SDS-PAGE theprotein was visualized by using Coomassie blue stain (GelCode, PierceCatalog #24596). Presence of the CIC was confirmed by using DNA-Silverstain (Pharmacia, Catalog #17-6000-30). Both SDS-PAGE and SEC-HPLC wereused to define pooling criteria, and for characterization of theobtained pool. Protein concentration was measured by the Bicinchoninicacid method (BCA, Sigma Catalog #BCA-1).

TABLE 21 Activity of C-11/Amb a 1 Conjugate in Mice IgG1 and IgG2aanti-Amb a 1 titers 2 weeks post 1st Imm 2 weeks post 2nd Imm GroupAnimal # Immunization IgG1 IgG2a IgG1 IgG2a 1 1 C-11/Amb a 1 30 148 7,900 19,886  2 conjugate 30 221  13,037 19,735  3 (1 ug) 30 943    94623,918  4 ID 30  64  5,485 10,487  5 38 1,894    3,805 9,945 6 30 943   600 5,249 7 30 570  10,337 20,156  8 30 259    600 8,350 9 56  30 2,575 5,747 10 30  30  8,381 28,971  mean  33** 510   5,367** 15,244*std dev  8 599  4,400 8,285 2 11 C-11/Amb a 1 51 345  8,982 27,877  12conjugate 30 667 201,008 612,739  13 (10 ug) 77 445  6,739 86,672  14 ID30 1,662    22,578 121,770  15 30  67 190,835 88,745  16 30 450  5,97117,600  17 55 1,137    29,646 105,398  18 99 1,119    70,159 183,152  1999 8,227    80,052 250,206  20 30 1,613    6,235 63,616  mean  53*1,573    62,221 155,778*  std dev 29 2,399    75,298 174,925  3 21P-6/Amb a 1 30  37  3,437 65,306  22 reference conjugate 1,422   303 15,652 6,198 23 (1 ug) 485  265  84,927 177,281  24 ID 170  1,182   37,379 56,074  25 903  2,027    38,121 76,572  26 88 2,298    32,499240,098  27 33 321  3,011 24,404  28 30  55  24,307 20,796  29 113   89 43,060 19,586  30 30  39  37,116 7,317 mean 330  662  31,951 69,363 std dev 475  862  23,568 78,697  4 31 Amb a 1 3,405   349 172,827 6,24432 (1 ug) 7,331    30 164,673 1,003 33 ID 2,847    35 112,766 7,174 344,021    30 100,281 1,399 35 8,333   212 156,037 4,969 36 1,214   286118,407 2,125 37 1,279    30 396,404   600 38 4,332    80 187,335 4,59939 569   30  63,536   600 40 2,696    30 161,039   902 mean 3,603**  111*  163,331**  2,962** std dev 2,554   123  90,406 2,530 a value of30 was used for samples <30 post 1st immunization a value of 600 wasused for samples <600 post 2nd immunization *p < 0.05, **p < 0.005compared to P-6/Amb a 1

TABLE 22

a value of 18 was used for values <18

**p < 0.005 compared P-6/Amb a 1 for 25 ug/ml restimulation

TABLE 23

a value of 24 was used for values <24

*p < 0.05, **p < 0.005 compared to P-6/Amb a 1 for 25 ug/mlrestimulation

Example 47 Effect of Spacer Moiety on CIC Activity

This example shows the effect of different spacer moieties on IFN-αinduction. Comparison of C-90 (C3 CIC) and C-51 (HEG CIC) showed thatC-51 induced 8-fold more IFN-α than C-90, although the amount of IFN-γinduced by each CIC was similar. Similarly, comparison of branched CICscontaining different linkers showed that for IFN-α induction, HEG(C-94)>TEG (C-103)>C3 (C-104)=no linker (C-28).

TABLE 24 IFN-γ (pg/ml) IFN-α (pg/ml) stim 28234 28235 28236 28237 mean28234 28235 28236 28237 mean cells alone 4 4 4 4 4 16 16 16 16 16 P-6 1552 51 1167 321 58 16 16 74 41 P-7 13 4 7 4 7 62 16 16 16 28 C-90 7 118497 1586 552 16 46 117 345 131 C-51 17 123 193 1580 478 16 77 352 37981061 C-71 30 168 448 1663 577 17 30 538 1665 563 C-101 14 205 627 2612865 16 249 1354 8566 2546 C-96 21 239 354 1396 503 16 120 608 993 434C-97 10 119 269 980 345 16 16 140 16 47 C-100 27 183 490 1907 652 16 16398 193 156 C-88 5 21 17 477 130 95 16 212 111 109 C-33 23 86 247 2076608 16 16 16 91 35 C-21 4 107 308 1645 516 16 16 73 678 196 C-28 10 1488 1229 335 16 16 16 16 16 C-94 7 161 239 1116 381 16 118 548 3631 1078C-103 21 44 250 1854 542 16 21 126 213 94 C-104 14 4 87 125 58 16 29 1616 19 PLGA 4 31 18 10 16 16 122 157 35 83 P-6 + PLGA 57 514 1052 37751350 16 694 1163 3444 1329 P-7 + PLGA 4 4 11 13 8 16 16 16 16 16 C-90 +PLGA 139 673 831 4618 1565 1175 696 4544 5103 2880 C-51 + PLGA 88 6441064 3748 1386 3257 2168 8000 8000 5356 C-71 + PLGA 101 797 1254 38991513 3085 2244 8000 8000 5332 C-101 + PLGA 110 659 879 6944 2148 46794488 8000 8000 6292 C-96 + PLGA 143 1070 1167 5471 1963 4107 3237 66608000 5501 C-97 + PLGA 68 737 988 5327 1780 4742 4216 8000 8000 6240C-100 + PLGA 176 1299 1742 7804 2755 1520 1092 4074 3777 2616 C-88 +PLGA 102 512 1148 5055 1704 803 613 2409 6412 2559 C-33 + PLGA 118 444968 3947 1369 551 566 3514 6727 2840 C-21 + PLGA 159 411 1089 4056 14291369 1561 5366 8000 4074 C-28 + PLGA 28 131 1005 3868 1258 16 16 184 13488 C-94 + PLGA 174 623 1352 4034 1546 4145 4653 7197 8000 5999 C-103 +PLGA 192 643 1388 5063 1822 895 1486 4456 5405 3061 C-104 + PLGA 40 73641 4930 1421 16 16 128 92 63 SAC 1845 1250 924 5350 2342 2374 327 11493744 1899

Example 48 Assessment of Isolated Immunomodulatory Activity ofPolynucleotides Corresponding in Sequence to CIC Nucleic Acid Moieties

This example further illustrates the immunostimulatory activity of CICsthat contain nucleic acid moieties that do not have isolatedimmunomodulatory activity. The activity of polynucleotides correspondingin sequence to the CIC nucleic acid moieties were assayed alone or incombination with free spacers, and compared to the activity of a CICcontaining the same amount of nucleic acid and spacer. For instance, 3uM of CIC C-101 was compared with 9 uM P-14 or a mixture of 9 uM P-14and 9 uM hexaethylene glycol and 3 uM glycerol (because C-101 containsthree equivalents of P-14, three equivalents of hexaethylene glycol, andone equivalent of glycerol.) In all cases, the CICs were active whilethe short polynucleotides, both alone and mixed with spacers, wereinactive. See Table 25. The activity of the spacers alone was tested ata concentration of 9 uM and all were completely inactive.

TABLE 25 IFN-g (pg/ml) IFN-a (pg/ml) stim 28250 28251 28252 28253 mean28250 28251 28252 28253 mean cells alone 18 5 9 1 8 16 16 30 19 20 P-6121 18 34 37 52 16 16 16 16 16 P-7 104 1 39 1 36 16 16 16 16 16 Propylspacer 13 1 1 1 4 16 16 16 16 16 Butyl spacer 8 5 1 1 4 16 16 16 16 16Triethylene glycol 15 1 7 1 6 16 16 16 16 16 Hexaethylene glycol 12 1 115 7 16 16 16 35 21 Glycerol 16 12 2 1 8 16 16 16 16 16 C-51 135 45 164167 128 181 246 95 1226 437 C-101 224 63 180 146 153 540 1472 509 26451291 P-14 10 34 11 10 16 16 16 16 16 16 P-14/HEG/glycerol 14 19 10 10 1316 16 16 16 16 C-21 122 51 155 203 133 31 69 41 264 101 C-94 340 60 287128 204 245 645 323 1198 603 P-1 54 21 56 1 33 16 16 16 16 16P-1/HEG/glycerol 15 9 19 1 11 16 16 16 16 16 C-45 107 26 95 8 59 16 10955 382 140 P-13 18 13 22 1 14 16 16 16 16 16 P-13/HEG 40 28 45 1 28 1616 16 16 16 C-10 337 163 776 898 544 16 23 25 124 47 P-2 7 25 53 1 22 1616 25 16 18 P-3 32 21 72 1 31 16 29 38 31 29 P-4 72 1 43 1 29 16 16 1616 16 P-2/P-3/P-4/HEG 68 1 38 1 27 16 16 16 16 16

Example 49 Preparation of (5′-TCGACGT-3′-HEG)_(ave=185)-Ficoll₄₀₀(C-137)

A. Preparation of Maleimido-Ficoll₄₀₀

Aminoethylcarboxymethyl (AECM)₁₈₀-Ficoll₄₀₀ was prepared by the methodof Inman (J. Immunology, 1975, 114: 704-709). On average there were 180aminoethyl groups per mole of Ficoll (MW=400,000 Da). 27.6 mg (62.6umol) of sulfosuccinimidyl4[N-maleimidomethyl]-cyclohexane-1-carboxylate dissolved in 300 ul ofDMSO was added dropwise, with constant vortexing, to 23.2 mg (0.058umol) of AECM₁₈₀-Ficoll₄₀₀ dissolved in 1.0 ml of 0.1 M sodium phosphatebuffer (pH 6.66). The reaction mixture was placed on a shaker for 2 hand then desalted on a Sephadex G-25 column to yield 20 mg ofmaleimido-Ficoll₄₀₀. On average, there were approximately 165 maleimidegroups per mole of Ficoll.

B. Preparation of 5′-TCGACGT-3′-HEG-(CH₂)₃—SH (C-136)

5′-TCGACGT-3′-HEG-(CH₂)₃—SS—(CH₂)₃—OH (C-135) was synthesizedanalogously to C-116. To 10 mg (3.57 umol) of C-135 dissolved in 0.4 mLof 0.1 M sodium phosphate/150 mM sodium chloride/pH 7.5 buffer was added5.7 mg (20 umol) of TCEP dissolved in 0.7 ml of the same buffer. Themixture was vortexed well and placed in a 40° C. water bath for 2 h. Thethiol (C-136) was purified by RP-HPLC (Polymer Labs PLRP-S column) usingan increasing gradient of acetonitrile in triethylammonium acetatebuffer (TEAA)/pH 7.0 and used immediately in the next reaction.

C. Preparation of (5′-TCGACGT-3′-HEG)_(x)-Ficoll₄₀₀ (C-137)

To 5.5 mg (0.014 umol) of maleimido-Ficoll₄₀₀ dissolved in 0.7 ml of 0.1M sodium phosphate/pH 6.66 was added 6.8 mg (2.5 umol) of C-136dissolved in 3.45 mL of approximately 30% acetonitrile/TEAA/pH 7.0buffer. The mixture was put on the shaker at RT overnight and theproduct was purified on a Superdex 200 column (Pharmacia). Calculationsusing the total weight of the isolated product and absorbance values at260 nm showed the product contained, on average, approximately 185oligonucleotides per mole of Ficoll. A second fraction containing alower loading of oligonucleotides per mole of Ficoll was also obtained.

D. Activity of C-137

As shown in Table 26, the polysaccaride based CIC had striking activityin the cytokine response assays, in particular showing significantstimulation of IFN-α.

TABLE 26 Compound IFN-g (pg/ml) IFN-a (pg/ml) stim 28313 28314 2831528316 mean ×4 28313 28314 28315 28316 mean cells alone 1 9 11 11 8 32 31122 100 98 88 P6 1 38 47 309 99 395 31 130 122 134 104 P7 1 11 12 20 1143 31 176 107 121 109 C-137 1 22 13 54 22 90 3612 5468 624 4000 3426 SAC87 77 56 4000 1055 4220 346 192 114 1172 456

Example 50 Synthesis of a CIC with a Branched Structure, Containing TwoDifferent 5′-Nucleic Acid Moieties

C-155, having the formula shown below, contains phosphorothioatelinkages in the nucleic acid moieties, between the nucleic acid moietiesand the HEG spacers, and between the HEG spacers and the glycerolbranching spacer.

C-155 was synthesized as described in Example 17, with the followingchanges: The instrument was programmed to add the nucleic acid moietiesand spacer moieties in the following order.

1. Use a 3′-support bound “A” solid support

2. Synthesis of 5′-TCGTCG-3′

3. Addition of HEG spacer phosphoramidite

4. Addition of asymmetrical branched phosphoramidite based on glycerol

5. Addition of HEG spacer phosphoramidite

6. Synthesis of 5′-TCGTCGA-3′

7. Detritylation and capping of the 5′-TCGTCGA-3′ moiety

8. Removal of the levulinyl protecting group

9. Addition of HEG spacer phosphoramidite

10. Synthesis of 5′-TCGACGT-3′

The CIC was purified by RP-HPLC as described in Example 12 andcharacterized as described in Example 2.

Example 51 Preparation of a Branched CIC with a Cage Structure UsingPhosphoramidite Chemistry

C-163, having the structure shown below and in FIG. 9F, is synthesizedas described in Example 20. All linkages are phosphorothioate.

C-163 (5′-CTGAACGTTCAG-3′-HEG)₃-trebler-HEG-5′- T-3′(CTGAACGTTCAG is SEQ ID NO:104). The three self-complimentary 12-mernucleic acid moieties are hybridized to a second molecule of the CIC, asshown in FIG. 9F, resulting in a cage structure. C-163, dissolved at aconcentration of approximately 1.0 mg/ml in 50 mM sodium phosphate/150mM sodium chloride/pH 7.2, is heated to 95° C. for 3 min and thenallowed to slowly cool in the heat block over a period of approximately2 hours. The formation of the cage structure is confirmed by sizeexclusion chromatography.

Example 52 Preparation of a Linear CIC with a Hairpin Structure

C-159, having the structure shown below, is synthesized as described inExample 2 and purified by RP-HPLC, as described in Example 12. Thelinkages in the nucleic acid moieties and between the nucleic acidmoieties and the HEG spacer are phosphorothioate.

C-159 5′-TGCGTGTAACGTTACACGCA-3′-HEG-5′- TGCGTGTAACGTTACACGCA-3′(TGCGTGTAACGTTACACGCA is SEQ ID NO:114). In C-159, the first nucleicacid moiety is complementary to the second nucleic acid moiety and formsa hairpin structure when annealed in the presence of salt, as describedin Example 51. C-160 is synthesized and annealed analogously.

Example 53 Preparation of a Branched CIC with a Central Spacer Structureusing a Conjugation Strategy

C-140 was synthesized as shown in FIG. 8G. To C-136 (7.3 mg, 2.7 umol),prepared as described in Example 49B, dissolved in 30% acetonitrile/0.1M triethyl ammonium acetate/pH 7.0 (4.0 mL) was added4-arm-bPEG-vinylsulfone (5.36 mg, 0.54 mmol, MW=10,000, ShearwaterPolymers, Inc., Huntsville, Ala.) dissolved in 100 mM sodiumphosphate/150 mM sodium chloride/pH 7.5 buffer (0.27 mL). The mixturewas placed on a circular mixer overnight at room temperature and thenpurified on a Superdex 200 column (Amersham Pharmacia, Piscataway, N.J.)using 10 mM sodium phosphate/150 mM sodium chloride/pH 7.2. C-139 wasprepared analogously.

Example 54 Preparation of Branched CICs with a Central Spacer Structure(C-168) and a Comb Structure (C-169) using Phosphoramidite Chemistry

The structures of C-168 and C-169 are shown below and in FIGS. 8D and8E. C-168 is synthesized as described in Example 17, with the followingchanges. C-168 contains phosphorothioate linkages in the nucleic acidmoieties, between the nucleic acid moieties and the HEG spacers, andbetween the HEG spacers and the glycerol branching spacer.

C-168 5′-TCGTCGA-3′-HEG-[gly(HEG-3′-TGCAGCT-5′)-HEG]₃- 5′-TCGAACG-3′The instrument is programmed to add the nucleic acid moieties and spacermoieties in the following order.

-   -   1. Use a 3′-support bound “G” solid support    -   2. Synthesis of 5′-TCGAAC-3′    -   3. Addition of HEG spacer phosphoramidite    -   4. Addition of asymmetrical spacer phosphoramidite based on        glycerol (gly)    -   5. Repeat steps 3 and 4 two more times    -   6. Addition of HEG spacer phosphoramidite    -   7. Synthesis of 5′-TCGTCGA-3′    -   8. Deprotect and cap the 5′-TCGTCGA-3′    -   9. Removal of the levulinyl protecting groups using a 90 min        treatment with 0.5 M hydrazine hydrate in pyridine:acetic acid        (1:1, v/v)    -   10. Addition of HEG spacer phosphoramidite    -   11. Synthesis of 5′-TCGACGT-3′        After removal of the 3 levulinyl protecting groups, as described        in Step 9, the reagents are added in amounts 2-3× the usual        amounts because three nucleic acid moieties are being        synthesized at one time. The CIC is purified by ion exchange        chromatography using Source Q 30 (Amersham Pharmacia,        Piscataway, N.J.) as described in Organic Process Research &        Development 2000, 4, 205-213.

C-169 is prepared analogously, except that Step 3′ is inserted betweenSteps 3 and 4, where Step 3′ is the synthesis of 5′-TTTTT-3′ and Step 5is the repetition of Steps 3′ and 4 two more times.

C-169 5′-TCGTCGA-3′-HEG-(gly(HEG-3′-TGCAGCT-5′)-5′-TTTTT-3′)₃-HEG-5′-TCGAACG-3′

Example 55 Preparation of a Self-Assembling CIC Containing aSelf-Complimentary Nucleic Acid Sequence That Can Form a Central AxisStructure

The structure of C-167 is shown in FIG. 9E and below. C-167 issynthesized as described in Example 19. C-167 contains phosphorothioatelinkages in the nucleic acid moieties, between the nucleic acid moietiesand the HEG spacers, and between the HEG spacers and the glycerolbranching spacer.

C-167 (5′-TCGACGT-3′-HEG)₂-glycerol-HEG-5′- TTGGCCAAGCTTGGCCAA-3′The self-complimentary 18-mer nucleic acid moiety in the CIC ishybridized to a second molecule of the CIC, as shown in FIG. 9E, bypreparing a solution of C-167 at a concentration of approximately 1.0mg/ml in 50 mM sodium phosphate/150 mM sodium chloride/pH 7.2, heatingthe solution to 95° C. for 3 mM, and then allowing the solution toslowly cool in the heat block over a period of approximately 2 hours.The formation of the double-stranded (central axis) CIC is confirmed bysize exclusion chromatography.

Example 56 Preparation of CICs with a Central Spacer Structure and anH-Structure (C-171) using Phosphoramidite Chemistry

The structures of C-171 and C-170 are shown below and in FIGS. 8F and8C. C-170 is synthesized as described in Example 17, with the followingchanges. C-170 contains phosphorothioate linkages in the nucleic acidmoieties, between the nucleic acid moieties and the HEG spacers, andbetween the HEG spacers and the glycerol branching spacer.

C-170 (5′-TCGACGT-3′-HEG)₂-glycerol-HEG-glycerol- (HEG-3′-TGCAGCT-5′)₂The instrument is programmed to add the nucleic acid moieties and spacermoieties in the following order.

-   -   1. Use a 5′-support bound “T” solid support    -   2. Synthesis of 3′-TGCAGC-5′ in the 5′ to 3′ direction (see        Example 14)    -   3. Addition of HEG spacer phosphoramidite    -   4. Addition of asymmetrical branched phosphoramidite based on        glycerol    -   5. Addition of HEG spacer phosphoramidite    -   6. Synthesis of 5′-TCGACGT-3′ in the 3′ to 5′ direction    -   7. Detritylation and capping of the 5′-TCGACGT-3′ moiety    -   8. Removal of the levulinyl protecting group with 0.5 M        hydrazine hydrate in pyridine:acetic acid (3:2, v/v), 5 min    -   9. Addition of HEG spacer phosphoramidite    -   10. Addition of symmetrical branched phosphoramidite based on        glycerol    -   11. Addition of HEG spacer phosphoramidite    -   12. Synthesis of 5′-TCGACGT-3′ in the 3′ to 5′ direction        For Steps 11 and 12, 2× the usual amount of reagents are used        because two chains are being synthesized simultaneously. This        method results in a CIC with an central spacer structure. A        second central spacer structure can be added to the first        central spacer structure by addition of a second asymmetric        branched phosphoramidite within one of the nucleic acid        moieties. If an asymmetric branched spacer is used in Step 10,        each nucleic acid moiety in the resulting CIC may contain a        different sequence.

C-171 is synthesized analogously, except that Step 9 is the synthesis of5′-TTTTT-3′ instead of addition of the HEG spacer phosphoramidite. C-170forms an H-structure.

C-171 (5′-TCGACGT-3′-HEG)₂-glycerol-5′-TTTTT-3′-glycerol-(HEG-3′-TGCAGCT-5′)₂

Example 57 Synthesis of Additional CICs

Additional compounds described in Table 2, supra have been synthesizedusing the following methods:

Com- pound Method of Synthesis C-138 as described in Example 49 C-141 asdescribed in Example 23 C-142, as described in Example 19 C-143, C-144,C-150, C-153, C-154, C-156, C-158 M-17- as described in Example 19,except that the appropriate M-20 spacers were inserted in place of thesymmetrical spacer and/or the HEG spacer C-151 and as described inExample 2, except that 6-amino-1-hexanol- M-21 CPG (AH-CPG; BioconjugateChem. 1992, 3, 85-87; Nucleic Acids Res. 1993, 21, 145-150) was used asthe solid support in order to generate a 3′-aminohexyl linker on the CICC-152 and as described in Example 19, except that 6-amino-1-hexanol-M-22 CPG (AH-CPG; Bioconjugate Chem. 1992, 3, 85-87; Nucleic Acids Res.1993, 21, 145-150) was used as the solid support in order to generate a3′-aminohexyl linker on the CIC

Additional compounds described in Table 2, supra, are synthesized usingthe following methods:

C-166 as described in Example 19, except that the linkages are oxidizedto phosphodiester linkages, as described in Example 25 C-159 and asdescribed in Example 2 C-160 C-163 as described in Example 20 C-164 asdescribed in Example 20, except that the linkages are oxidized tophosphodiester linkages, as described in Example 25 C-161, C-162, asdescribed in Example 19 C-165

Compounds M-17-M-22, among others described herein, do not include a CGmotif and are used generally as controls in assays or experiments.

The remaining CICs and oligonucleotides (e.g., C-172, C-173-176, C-177,P-17, C-178, C-179-184, C-185-187, C-188-197, C-198-203, C-204-207;C-208-209, and M2-3) were synthesized analogously to those describedherein.

CIC duplexes (e.g., C-202/C-203 duplex; C-208/C-209 duplex; C-202/C-209duplex; C-203/C-208 duplex; and C-178 homoduplex) were prepared byannealing (1 mg/ml oligonucleotide heated 5 m at 95° C. in PBS, allowedto cool slowly to room temperature, and stored at 4° C.).

Example 58 Induction of IFN-α Secretion by Multimeric CICs

The ability of CICs and oligonucleotides to elicit IFN-α from humanPBMCs was assayed as described in Example 28. The results shown belowdemonstrate that CICs that can self-hybridize (C-173, C-174, C-175)induce significantly more IFN-α from human PBMC than does the parentoligonucleotide (P-17) when used at low doses (e.g., 0.8 ug/ml). Eachcompound was assayed at three concentrations using PBMCs from fourdifferent individuals and the mean values determined.

IFN-α (pg/ml) Stim (amt, ug/ml) 1 2 3 4 mean medium 52 52 52 52 52 P-6(20) 116 64 52 52 71 P-6 (4) 115 52 52 65 71 P-6 (0.8) 52 52 52 52 52P-7 (20) 52 52 52 52 52 P-7 (4) 52 52 52 52 52 P-7 (0.8) 52 52 52 52 52C-101 (20) 2330 109 215 157 703 C-101 (4) 52 52 59 204 92 C-101 (0.8) 5252 52 52 52 M-3 (20) 52 52 52 52 52 M-3 (4) 52 52 52 52 52 M-3 (0.8) 5252 52 52 52 C-173 (20) 14381 8060 66 299 5701 C-173 (4) 3828 2917 21973563 3126 C-173 (0.8) 272 1293 1138 2547 1312 C-174 (20) 1350 1176 61837 856 C-174 (4) 5601 7845 1016 5895 5089 C-174 (0.8) 7907 11198 9981752 5464 C-175 (20) 2250 732 52 52 771 C-175 (4) 7134 6779 906 19514193 C-175 (0.8) 21783 16605 851 2874 10528 P-17 (20) 1022 1792 52 52729 P-17 (4) 9154 10388 531 837 5228 P-17 (0.8) 52 105 52 286 124

Example 59 Induction of IFN-α Secretion by Multimeric CICs

Assays were conducted as described for Example 58. The results showndemonstrate that CICs that hybridize to produce multimers with a totalof four free 5′-ends with active TCG-containing heptamers (e.g., C-178duplex, C-202/C-203 heteroduplex) induce significantly more IFN-α fromhuman PBMC than CIC multimers containing only two free 5′-ends (C-101,C-202, C-203).

IFN-α (pg/ml) stim 48 177 272 273 mean medium 102 102 102 102 102 P-6(20) 256 323 102 102 196 P-6 (4) 102 245 102 102 138 P-6 (0.8) 102 102102 102 102 P-7 (20) 102 102 102 102 102 P-7 (4) 102 102 102 102 102 P-7(0.8) 102 102 102 102 102 C-101 (20) 2212 4277 642 102 1808 C-101 (4)818 3631 604 102 1289 C-101 (0.8) 102 102 185 102 123 M-3(20) 102 102102 102 102 M-3 (4) 102 102 102 102 102 M-3 (0.8) 102 102 102 102 102C-178 (20) 13946 19973 15050 3971 13235 C-178 (4) 16300 8304 1677 17327003 C-178 (0.8) 336 1150 149 102 434 C-202 (20) 1901 2742 612 339 1399C-202 (4) 2250 2482 1222 215 1542 C-202 (0.8) 830 102 102 102 284 C-203(20) 735 681 102 102 405 C-203 (4) 691 487 102 102 346 C-203 (0.8) 102102 102 102 102 C-202/C-203 (20) 18550 17237 3474 2285 10386 C-202/C-203(4) 18550 17237 14509 6717 14253 C-202/C-203 (0.8) 3399 1545 688 1021434

***

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced. Therefore,descriptions and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

All patents, patent applications, and publications cited herein arehereby incorporated by reference in their entirety for all purposes tothe same extent as if each individual publication, patent or patentapplication were specifically and individually indicated to be soincorporated by reference.

We claim:
 1. A linear chimeric immunostimulatory compound (CIC)comprising the structure N₁-S₁-N₂-S₂-N₃, wherein N₁, N₂, and N₃ areindependently selected nucleic acid moieties, at least one of N₁, N₂,and N₃ has the sequence 5′-TCGY-3′, where Y is selected from the groupconsisting of XCGX, XTCG, XXCG, and CGXX, where each X is anindependently selected nucleotide; wherein S₁ is a non-nucleic acidspacer moiety covalently bound to N₁ and N₂; S₂ is a non-nucleic acidspacer moiety covalently bound to N₂ and N₃; S₁ and S₂ are the same ordifferent; each of S ₁ and S₂ comprises hexaethylene glycol (HEG),triethylene glycol (TEG), propyl, butyl, or hexyl; and wherein said CIChas at least one immunostimulatory activity selected from the groupconsisting of (i) the ability to stimulate interferon-gamma (IFN-γ)production by human peripheral blood mononuclear cells or (ii) theability to stimulate interferon-alpha (IFN-α) production by humanperipheral blood mononuclear cells.
 2. The CIC of claim 1 wherein atleast one of S₁ and S₂ is a HEG spacer moiety.
 3. The CIC of claim 2wherein both S₁ and S₂ are HEG spacer moieties.
 4. The CIC of claim 1wherein at least one of N₁ and N₃ has a sequence 5′-TCGXCGX-3′.
 5. TheCIC of claim 4 wherein at least one nucleic acid moiety comprises thesequence 5′-TCGCCGG-3′, 5′-TCGGCGC-3′ or 5′-TCGTCGT-3′.
 6. The CIC ofclaim 1 wherein at least one of N₁ and N₂ has a sequence 5′-TCGXCGX-3′.7. The CIC of claim 6 wherein at least one nucleic acid moiety comprisesthe sequence 5′-TCGCCGG-3′, 5′-TCGGCGC-3′ or 5′-TCGTCGT-3′.
 8. The CICof claim 1 wherein at least one of N₂ and N₃ has a sequence5′-[(X)₀₋₂]TCG[(X)₂₋₄]-3′, wherein the first X at the 5′ end of the CICis an A and each following X is an independently selected nucleotide. 9.A linear chimeric immunostimulatory compound (CIC) comprising thestructure N₁-S₁-N₂-S₂-N₃, wherein N₁, N₂, and N₃ are independentlyselected nucleic acid moieties and wherein at least one nucleic acidmoiety comprises the sequence 5′-AACGTTC-3′, 5′-AACGTT-3′, 5′-ATCGT-3′,5′-ATCGAT-3′, 5′-ACCGGT-3′, 5′-AGCGTT-3′, 5′-ATCGTT-3′, 5′-AACGAT-3′,5′-AACGCT-3′, 5′-AACGTG-3′, 5′-AACGTA-3′, or 5′-AACGTC-3′, wherein S₁ isa non-nucleic acid spacer moiety covalently bound to N₁ and N₂; S₂ is anon-nucleic acid spacer moiety covalently bound to N₂ and N₃; S₁ and S₂are the same or different; each of S₁ and S₂ comprises hexaethyleneglycol (HEG), triethylene glycol (TEG), propyl, butyl, or hexyl; andwherein said CIC has at least one immunostimulatory activity selectedfrom the group consisting of (i) the ability to stimulateinterferon-gamma (IFN-γ) production by human peripheral bloodmononuclear cells or (ii) the ability to stimulate interferon-alpha(IFN-α) production by human peripheral blood mononuclear cells.
 10. TheCIC of claim 9 wherein at least one nucleic acid moiety comprises thesequence 5′-AACGTTC-3′.
 11. The CIC of claim 1, where the linkagesbetween the nucleic acid moieties and the nonnucleic acid spacermoieties comprise one or more linkages selected from the groupconsisting of phosphodiester linkages, phosphorothioate ester linkages,and phosphorodithioate ester linkages.
 12. A composition comprising aCIC of claim 1 and a pharmaceutically acceptable excipient.
 13. Acomposition comprising a CIC of claim 1 and further comprising anantigen.
 14. A composition comprising a CIC of claim 1 and furthercomprising a cationic microsphere.
 15. The composition of claim 14wherein the microsphere comprises a polymer of lactic acid and glycolicacid.
 16. A CIC of claim 1 comprising a reactive linking group.
 17. ACIC of claim 16 comprising a reactive thio group.
 18. A CIC of claim 1linked to a polypeptide.